Peptidomimetic macrocycles

ABSTRACT

The present invention provides novel peptidomimetic macrocycles and methods of using such macrocycles for the treatment of disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. Nos. 61/373,701 filed Aug. 13, 2010, 61/373,638 filedAug. 13, 2010, and 61/374,163 filed Aug. 16, 2010, which are herebyincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The human transcription factor protein p53 induces cell cycle arrest andapoptosis in response to DNA damage and cellular stress, and therebyplays a critical role in protecting cells from malignant transformation.The E3 ubiquitin ligase HDM2 negatively regulates p53 function through adirect binding interaction that neutralizes the p53 transactivationactivity, leads to export from the nucleus of p53 protein, and targetsp53 for degradation via the ubiquitylation-proteasomal pathway. Loss ofp53 activity, either by deletion, mutation, or HDM2 overexpression, isthe most common defect in human cancers. Tumors that express wild typep53 are vulnerable to pharmacologic agents that stabilize or increasethe concentration of active p53. In this context, inhibition of theactivities of HDM2 has emerged as a validated approach to restore p53activity and resensitize cancer cells to apoptosis in vitro and in vivo.HDMX (HDM4) has more recently been identified as a similar negativeregulator of p53, and studies have revealed significant structuralhomology between the p53 binding interfaces of HDM2 and HDMX.

The p53-HDM2 and p53-HDMX protein-protein interactions are mediated bythe same 15-residue alpha-helical transactivation domain of p53, whichinserts into hydrophobic clefts on the surface of HDM2 and HDMX. Threeresidues within this domain of p53 (F19, W23, and L26) are essential forbinding to HDM2 and HDMX. The present invention provides p53-basedpeptidomimetic macrocycles that modulate the activities of p53 byinhibiting the interactions between p53 and HDM2, p53 and HDMX, or p53and both HDM2 and HDMX proteins, and that may be used for treatingdiseases including but not limited to cancer and otherhyperproliferative diseases.

SUMMARY OF THE INVENTION

Described below are stably cross-linked peptides related to a portion ofhuman p53 (“p53 peptidomimetic macrocycles”). These cross-linkedpeptides contain at least two modified amino acids that together form anintramolecular cross-link that can help to stabilize the alpha-helicalsecondary structure of a portion of p53 that is thought to be importantfor binding of p53 to HDM2 and for binding of p53 to HDMX. Accordingly,a cross-linked polypeptide described herein can have improved biologicalactivity relative to a corresponding polypeptide that is notcross-linked. The p53 peptidomimetic macrocycles are thought tointerfere with binding of p53 to HDM2 and/or of p53 to HDMX, therebyliberating functional p53 and inhibiting its destruction. The p53peptidomimetic macrocycles described herein can be used therapeutically,for example to treat cancers and other disorders characterized by anundesirably low level or a low activity of p53, and/or to treat cancersand other disorders characterized by an undesirably high level ofactivity of HDM2 or HDMX. The p53 peptidomimetic macrocycles may also beuseful for treatment of any disorder associated with disruptedregulation of the p53 transcriptional pathway, leading to conditions ofexcess cell survival and proliferation such as cancer and autoimmunity,in addition to conditions of inappropriate cell cycle arrest andapoptosis such as neurodegeneration and immunedeficiencies. In someinstances, the p53 peptidomimetic macrocycles bind to HDM2 (e.g.,GenBank® Accession No.: 228952; GI:228952) and/or HDMX (also referred toas HDM4; GenBank® Accession No.: 88702791; GI:88702791).

In one aspect, the present invention provides a peptidomimeticmacrocycle comprising an amino acid sequence which is at least about60%, 80%, 90%, or 95% identical to an amino acid sequence chosen fromthe group consisting of the amino acid sequences in Table 1, 2, 3, or 4.Alternatively, an amino acid sequence of said peptidomimetic macrocycleis chosen from the group consisting of the amino acid sequences inTable 1. Alternatively, an amino acid sequence of said peptidomimeticmacrocycle is chosen as above, and further wherein the macrocycle doesnot include a thioether or a triazole. In some embodiments, thepeptidomimetic macrocycle comprises a helix, such as an α-helix. Inother embodiments, the peptidomimetic macrocycle comprises anα,α-disubstituted amino acid. A peptidomimetic macrocycle of theinvention may comprise a crosslinker linking the α-positions of at leasttwo amino acids. At least one of said two amino acids may be anα,α-disubstituted amino acid.

In some embodiments, the peptidomimetic macrocycle has the formula:

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid, and the terminal D and E independently optionally include acapping group;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO-], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v and w are independently integers from 1-1000;

u is an integer from 1-10;

x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

In various embodiments, the peptidomimetic macrocycle includes L₁ and L₂wherein L₁ and L₂ either alone or in combination do not include athioether or a triazole.

In other embodiments, the peptidomimetic macrocycle may comprise acrosslinker linking a backbone amino group of a first amino acid to asecond amino acid within the peptidomimetic macrocycle. For example, theinvention provides peptidomimetic macrocycles of the formula (IV) or(IVa):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid, and the terminal D and E independently optionally include acapping group;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO-], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

v and w are independently integers from 1-1000;

u is an integer from 1-10;

x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

Additionally, the invention provides a method of treating cancer in asubject comprising administering to the subject a peptidomimeticmacrocycle of the invention. Also provided is a method of modulating theactivity of p53 or HDM2 or HDMX in a subject comprising administering tothe subject a peptidomimetic macrocycle of the invention, or a method ofantagonizing the interaction between p53 and HDM2 and/or HDMX proteinsin a subject comprising administering to the subject such apeptidomimetic macrocycle.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 describes the synthesis of Fmoc-Me-6-Chloro-Tryptophan &Fmoc-6-Chloro-Tryptophan.

FIG. 2 shows an LC-MS trace of Me-6-Chloro-(Boc)Tryptophan-Ni—S—BPB.

FIG. 3 shows a 1H-NMR spectrum of Me-6-Chloro-(Boc)Tryptophan-Ni—S—BPB.

FIG. 4 shows an LC-MS trace of Fmoc-Me-6-Chloro-(Boc)Tryptophan.

FIG. 5 shows a 1H-NMR spectrum of Fmoc-Me-6-Chloro-(Boc)Tryptophan.

FIGS. 6 a-f describe the results of a cell viability assay, acompetition ELISA assay, GRIP assay, Kd data, p21 activation assay,fluorescence polarization competition binding and circular helicity datafor exemplary peptidomimetic macrocycles of the invention.

FIGS. 7A-D provide data from a variety of macrocycles.

FIGS. 8A-B provide data from a variety of macrocycles.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “macrocycle” refers to a molecule having achemical structure including a ring or cycle formed by at least 9covalently bonded atoms.

As used herein, the term “peptidomimetic macrocycle” or “crosslinkedpolypeptide” refers to a compound comprising a plurality of amino acidresidues joined by a plurality of peptide bonds and at least onemacrocycle-forming linker which forms a macrocycle between a firstnaturally-occurring or non-naturally-occurring amino acid residue (oranalog) and a second naturally-occurring or non-naturally-occurringamino acid residue (or analog) within the same molecule. Peptidomimeticmacrocycle include embodiments where the macrocycle-forming linkerconnects the α carbon of the first amino acid residue (or analog) to theα carbon of the second amino acid residue (or analog). Thepeptidomimetic macrocycles optionally include one or more non-peptidebonds between one or more amino acid residues and/or amino acid analogresidues, and optionally include one or more non-naturally-occurringamino acid residues or amino acid analog residues in addition to anywhich form the macrocycle. A “corresponding uncrosslinked polypeptide”when referred to in the context of a peptidomimetic macrocycle isunderstood to relate to a polypeptide of the same length as themacrocycle and comprising the equivalent natural amino acids of thewild-type sequence corresponding to the macrocycle.

As used herein, the term “stability” refers to the maintenance of adefined secondary structure in solution by a peptidomimetic macrocycleof the invention as measured by circular dichroism, NMR or anotherbiophysical measure, or resistance to proteolytic degradation in vitroor in vivo. Non-limiting examples of secondary structures contemplatedin this invention are α-helices, β-turns, and β-pleated sheets.

As used herein, the term “helical stability” refers to the maintenanceof a helical structure by a peptidomimetic macrocycle of the inventionas measured by circular dichroism or NMR. For example, in someembodiments, the peptidomimetic macrocycles of the invention exhibit atleast a 1.25, 1.5, 1.75 or 2-fold increase in α-helicity as determinedby circular dichroism compared to a corresponding uncrosslinkedmacrocycle.

The term “α-amino acid” or simply “amino acid” refers to a moleculecontaining both an amino group and a carboxyl group bound to α carbonwhich is designated the α-carbon. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. Unless the contextspecifically indicates otherwise, the term amino acid, as used herein,is intended to include amino acid analogs.

The term “naturally occurring amino acid” refers to any one of thetwenty amino acids commonly found in peptides synthesized in nature, andknown by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L,K, M, F, P, S, T, W, Y and V.

The term “amino acid analog” or “non-natural amino acid” refers to amolecule which is structurally similar to an amino acid and which can besubstituted for an amino acid in the formation of a peptidomimeticmacrocycle. Amino acid analogs include, without limitation, compoundswhich are structurally identical to an amino acid, as defined herein,except for the inclusion of one or more additional methylene groupsbetween the amino and carboxyl group (e.g., α-amino β-carboxy acids), orfor the substitution of the amino or carboxy group by a similarlyreactive group (e.g., substitution of the primary amine with a secondaryor tertiary amine, or substitution of the carboxy group with an ester).Non-natural amino acids include structures according to the following:

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide without abolishing orsubstantially altering its essential biological or biochemical activity(e.g., receptor binding or activation). An “essential” amino acidresidue is a residue that, when altered from the wild-type sequence ofthe polypeptide, results in abolishing or substantially abolishing thepolypeptide's essential biological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains(e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V,I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a polypeptide, for example, ispreferably replaced with another amino acid residue from the same sidechain family. Other examples of acceptable substitutions aresubstitutions based on isosteric considerations (e.g. norleucine formethionine) or other properties (e.g. 2-thienylalanine forphenylalanine).

The term “capping group” refers to the chemical moiety occurring ateither the carboxy or amino terminus of the polypeptide chain of thesubject peptidomimetic macrocycle. The capping group of a carboxyterminus includes an unmodified carboxylic acid (ie —COOH) or acarboxylic acid with a substituent. For example, the carboxy terminusmay be substituted with an amino group to yield a carboxamide at theC-terminus. Various substituents include but are not limited to primaryand secondary amines, including pegylated secondary amines.Representative secondary amine capping groups for the C-terminusinclude:

The capping group of an amino terminus includes an unmodified amine (ie—NH₂) or an amine with a substituent. For example, the amino terminusmay be substituted with an acyl group to yield a carboxamide at theN-terminus. Various substituents include but are not limited tosubstituted acyl groups, including C₁-C₆ carbonyls, C₇-C₃₀ carbonyls,and pegylated carbamates. Representative capping groups for theN-terminus include:

The term “member” as used herein in conjunction with macrocycles ormacrocycle-forming linkers refers to the atoms that form or can form themacrocycle, and excludes substituent or side chain atoms. By analogy,cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are allconsidered ten-membered macrocycles as the hydrogen or fluorosubstituents or methyl side chains do not participate in forming themacrocycle.

The symbol “

” when used as part of a molecular structure refers to a single bond ora trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acid. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for aspartate is carboxymethyl, the amino acidside chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included, forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an α,α di-substituted aminoacid).

The term “α,α di-substituted amino” acid refers to a molecule or moietycontaining both an amino group and a carboxyl group bound to α carbon(the α-carbon) that is attached to two natural or non-natural amino acidside chains.

The term “polypeptide” encompasses two or more naturally ornon-naturally-occurring amino acids joined by a covalent bond (e.g., anamide bond). Polypeptides as described herein include full lengthproteins (e.g., fully processed proteins) as well as shorter amino acidsequences (e.g., fragments of naturally-occurring proteins or syntheticpolypeptide fragments).

The term “macrocyclization reagent” or “macrocycle-forming reagent” asused herein refers to any reagent which may be used to prepare apeptidomimetic macrocycle of the invention by mediating the reactionbetween two reactive groups. Reactive groups may be, for example, anazide and alkyne, in which case macrocyclization reagents include,without limitation, Cu reagents such as reagents which provide areactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II)salts such as Cu(CO₂CH₃)₂, CuSO₄, and CuCl₂ that can be converted insitu to an active Cu(I) reagent by the addition of a reducing agent suchas ascorbic acid or sodium ascorbate. Macrocyclization reagents mayadditionally include, for example, Ru reagents known in the art such asCp*RuCl(PPh₃)₂, [Cp*RuCl]₄ or other Ru reagents which may provide areactive Ru(II) species. In other cases, the reactive groups areterminal olefins. In such embodiments, the macrocyclization reagents ormacrocycle-forming reagents are metathesis catalysts including, but notlimited to, stabilized, late transition metal carbene complex catalystssuch as Group VIII transition metal carbene catalysts. For example, suchcatalysts are Ru and Os metal centers having a +2 oxidation state, anelectron count of 16 and pentacoordinated. Additional catalysts aredisclosed in Grubbs et al., “Ring Closing Metathesis and RelatedProcesses in Organic Synthesis” Acc. Chem. Res. 1995, 28, 446-452, andU.S. Pat. No. 5,811,515. In yet other cases, the reactive groups arethiol groups. In such embodiments, the macrocyclization reagent is, forexample, a linker functionalized with two thiol-reactive groups such ashalogen groups.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine oriodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chainor branched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive)carbon atoms in it. In the absence of any numerical designation, “alkyl”is a chain (straight or branched) having 1 to 20 (inclusive) carbonatoms in it.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenylchain. In the absence of any numerical designation, “alkenyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₆ alkynylchain. In the absence of any numerical designation, “alkynyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring aresubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkylgroup, as defined above. Representative examples of an arylalkyl groupinclude, but are not limited to, 2-methylphenyl, 3-methylphenyl,4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl,2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl,3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl,4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl,3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyland 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with one or more—C(O)NH₂ groups. Representative examples of an arylamido group include2-C(O)NH2-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl,3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl,

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above,wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replacedwith a heterocycle. Representative examples of an alkylheterocycle groupinclude, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine,—CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—C(O)NH₂ group. Representative examples of an alkylamido group include,but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂,—CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂,—CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃,—C(CH₃)₂CH₂C(O)NH₂, —CH₂—CH₂—NH—C(O)—CH₃, —CH₂—CH₂—NH—C(O)—CH₃—CH3, and—CH₂—CH₂—NH—C(O)—CH═CH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein oneof the C₁-C₅ alkyl group's hydrogen atoms has been replaced with ahydroxyl group. Representative examples of an alkanol group include, butare not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂ CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and—C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced witha—COOH group. Representative examples of an alkylcarboxy group include,but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH,—CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH,—CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally is optionally substituted. Somecycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring are substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituent” refers to a group replacing a second atom orgroup such as a hydrogen atom on any molecule, compound or moiety.Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds of this invention contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are included in thepresent invention unless expressly provided otherwise. In someembodiments, the compounds of this invention are also represented inmultiple tautomeric forms, in such instances, the invention includes alltautomeric forms of the compounds described herein (e.g., if alkylationof a ring system results in alkylation at multiple sites, the inventionincludes all such reaction products). All such isomeric forms of suchcompounds are included in the present invention unless expresslyprovided otherwise. All crystal forms of the compounds described hereinare included in the present invention unless expressly providedotherwise.

As used herein, the terms “increase” and “decrease” mean, respectively,to cause a statistically significantly (i.e., p<0.1) increase ordecrease of at least 5%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable is equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variableis equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 takes thevalues 0, 1 or 2 if the variable is inherently discrete, and takes thevalues 0.0, 0.1, 0.01, 0.001, or any other real values ≧0 and ≦2 if thevariable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The term “on average” represents the mean value derived from performingat least three independent replicates for each data point.

The term “biological activity” encompasses structural and functionalproperties of a macrocycle of the invention. Biological activity is, forexample, structural stability, alpha-helicity, affinity for a target,resistance to proteolytic degradation, cell penetrability, intracellularstability, in vivo stability, or any combination thereof.

The details of one or more particular embodiments of the invention areset forth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

In some embodiments, the peptide sequences are derived from the p53protein.

A non-limiting exemplary list of suitable p53 peptides for use in thepresent invention is given below.

TABLE 1 Sequence (bold =critical residue;  X  = cross-linked amino acid)Design Notes Ac- Gln Ser Gln Gln Thr Phe Ser Asn Leu Trp Arg Leu Leu ProGln Asn —NH2 linear Ac- X Gln Ser Gln X Thr Phe Ser Asn Leu Trp Arg LeuLeu Pro Gln Asn —NH2 i--> i + 4 x-link #1 Ac- X Ser Gln Gln X Phe SerAsn Leu Trp Arg Leu Leu Pro Gln Asn —NH2 i--> i + 4 x-link #2 Ac- GlnSer X Gln Thr Phe X Asn Leu Trp Arg Leu Leu Pro Gln Asn —NH2 i--> i +4 x-link #3 Ac- Gln Ser Gln X Thr Phe Ser X Leu Trp Arg Leu Leu Pro GlnAsn —NH2 i--> i + 4 x-link #4 Ac- Gln Ser Gln Gln X Phe Ser Asn X TrpArg Leu Leu Pro Gln Asn —NH2 i--> i + 4 x-link #5 Ac- Gln Ser Gln GlnThr Phe X Asn Leu Trp X Leu Leu Pro Gln Asn —NH2 i--> i + 4 x-link #6Ac- Gln Ser Gln Gln Thr Phe Ser X Leu Trp Arg X Leu Pro Gln Asn —NH2i--> i + 4 x-link #7 Ac- Gln Ser Gln Gln Thr Phe Ser Asn Leu Trp X LeuLeu Pro X Asn —NH2 i--> i + 4 x-link #8 Ac- Gln Ser Gln Gln Thr Phe SerAsn Leu Trp Arg X Leu Pro Gln X —NH2 i--> i + 4 x-link #9 Ac- X Gln SerGln Gln Thr Phe X Asn Leu Trp Arg Leu Leu Pro Gln Asn —NH2 i--> i +7 x-link #1 Ac- X Ser Gln Gln Thr Phe Ser X Leu Trp Arg Leu Leu Pro GlnAsn —NH2 i--> i + 7 x-link #2 Ac- Gln X Gln Gln Thr Phe Ser Asn X TrpArg Leu Leu Pro Gln Asn —NH2 i--> i + 7 x-link #3 Ac- Gln Ser Gln X ThrPhe Ser Asn Leu Trp X Leu Leu Pro Gln Asn —NH2 i--> i + 7 x-link #4 Ac-Gln Ser Gln Gln X Phe Ser Asn Leu Trp Arg X Leu Pro Gln Asn —NH2 i-->i + 7 x-link #5 Ac- Gln Ser Gln Gln Thr Phe X Asn Leu Trp Arg Leu Leu XGln Asn —NH2 i--> i + 7 x-link #6 Ac- Gln Ser Gln Gln Thr Phe Ser X LeuTrp Arg Leu Leu Pro X Asn —NH2 i--> i + 7 x-link #7 Ac- Gln Ser Gln GlnThr Phe Ser Asn X Trp Arg Leu Leu Pro Gln X —NH2 i--> i + 7 x-link #8

TABLE 2 Sequence (bold = critical residue;  X  =cross-linked amino acid) Design Notes Ac- Leu Thr Phe Glu His Tyr TrpAla Gln Leu Thr Ser —NH2 linear Ac- X Leu Thr Phe X His Tyr Trp Ala GlnLeu Thr Ser —NH2 i--> i + 4 x-link #1 Ac- X Thr Phe Glu X Tyr Trp AlaGln Leu Thr Ser —NH2 i--> i + 4 x-link #2 Ac- Leu X Phe Glu His X TrpAla Gln Leu Thr Ser —NH2 i--> i + 4 x-link #3 Ac- Leu Thr Phe X His TyrTrp X Gln Leu Thr Ser —NH2 i--> i + 4 x-link #4 Ac- Leu Thr Phe Glu XTyr Trp Ala X Leu Thr Ser —NH2 i--> i + 4 x-link #5 Ac- Leu Thr Phe GluHis Tyr Trp X Gln Leu Thr X —NH2 i--> i + 4 x-link #6 Ac- Leu Thr PheGlu His Tyr Trp Ala X Leu Thr Ser X —NH2 i--> i + 4 x-link #7 Ac- X ThrPhe Glu His Tyr Trp X Gln Leu Thr Ser —NH2 i--> i + 7 x-link #1 Ac- GlnX Phe Glu His Tyr Trp Ala X Leu Thr Ser —NH2 i--> i + 7 x-link #2 Ac-Gln Thr Phe X His Tyr Trp Ala Gln Leu X Ser —NH2 i--> i + 7 x-link #3Ac- Gln Thr Phe Glu X Tyr Trp Ala Gln Leu Thr X —NH2 i--> i + 7 x-link#4 Ac- Gln Thr Phe Glu His X Trp Ala Gln Leu Thr Ser X —NH2 i--> i +7 x-link #5

TABLE 3 Sequence bold = critical residue;  X  = cross-linked amino acidDesign Notes Ac- Phe Met Aib/His/Asn Tyr 6-ClTrp Glu Ac3c/Gln/Leu Leu—NH2 linear Ac- X Phe Met Aib/His/Asn X 6-ClTrp Glu Ac3c/Gln/Leu Leu—NH2 i--> i + 4 x-link #1 Ac- Phe X Aib/His/Asn Tyr 6-ClTrp XAc3c/Gln/Leu Leu —NH2 i--> i + 4 x-link #2 Ac- Phe Met X Tyr 6-ClTrp GluX Leu —NH2 i--> i + 4 x-link #3 Ac- X Phe Met Aib/His/Asn Tyr 6-ClTrpGlu X Leu —NH2 i--> i + 7 x-link #1 Ac- Phe X Aib/His/Asn Tyr 6-ClTrpGlu Ac3c/Gln/Leu Leu X —NH2 i--> i + 7 x-link #2

In Table 3 and elsewhere, “Aib” represents a 2-aminoisobutyric acidresidue, while “Ac3c” represents a aminocyclopropane carboxylic acidresidue.

Peptidomimetic Macrocycles

In some embodiments, a peptidomimetic macrocycle of the invention hasthe Formula (I):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid, and the terminal D and E independently optionally include acapping group;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO-], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v and w are independently integers from 1-1000;

u is an integer from 1-10;

x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

In one embodiment, L₁ and L₂, either alone or in combination, do notform a triazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A], when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges. Similarly, when u is greater than 1, each compound of theinvention may encompass peptidomimetic macrocycles which are the same ordifferent. For example, a compound of the invention may comprisepeptidomimetic macrocycles comprising different linker lengths orchemical compositions.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

In one embodiment, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁ and R₂ is independently independently —H, alkyl,alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl,or heterocycloalkyl, unsubstituted or substituted with halo-.

In related embodiments, the peptidomimetic macrocycle of Formula (I) is:

In other embodiments, the peptidomimetic macrocycle of Formula (I) is acompound of any of the formulas shown below:

wherein “AA” represents any natural or non-natural amino acid side chainand “

” is [D]_(v), [E]_(w) as defined above, and n is an integer between 0and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. Inother embodiments, n is less than 50.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

In other embodiments, D and/or E in the compound of Formula I arefurther modified in order to facilitate cellular uptake. In someembodiments, lipidating or PEGylating a peptidomimetic macrocyclefacilitates cellular uptake, increases bioavailability, increases bloodcirculation, alters pharmacokinetics, decreases immunogenicity and/ordecreases the needed frequency of administration.

In other embodiments, at least one of [D] and [E] in the compound ofFormula I represents a moiety comprising an additionalmacrocycle-forming linker such that the peptidomimetic macrocyclecomprises at least two macrocycle-forming linkers. In a specificembodiment, a peptidomimetic macrocycle comprises two macrocycle-forminglinkers. In an embodiment, u is 2.

In the peptidomimetic macrocycles of the invention, any of themacrocycle-forming linkers described herein may be used in anycombination with any of the sequences shown in Tables 1-4 and also withany of the R-substituents indicated herein.

In some embodiments, the peptidomimetic macrocycle comprises at leastone α-helix motif For example, A, B and/or C in the compound of FormulaI include one or more α-helices. As a general matter, α-helices includebetween 3 and 4 amino acid residues per turn. In some embodiments, theα-helix of the peptidomimetic macrocycle includes 1 to 5 turns and,therefore, 3 to 20 amino acid residues. In specific embodiments, theα-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In someembodiments, the macrocycle-forming linker stabilizes an α-helix motifincluded within the peptidomimetic macrocycle. Thus, in someembodiments, the length of the macrocycle-forming linker L from a firstCα to a second Cα is selected to increase the stability of an α-helix.In some embodiments, the macrocycle-forming linker spans from 1 turn to5 turns of the α-helix. In some embodiments, the macrocycle-forminglinker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turnsof the α-helix. In some embodiments, the length of themacrocycle-forming linker is approximately 5 Å to 9 Å per turn of theα-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelength is equal to approximately 5 carbon-carbon bonds to 13carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 2 turns of an α-helix, thelength is equal to approximately 8 carbon-carbon bonds to 16carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 3 turns of an α-helix, thelength is equal to approximately 14 carbon-carbon bonds to 22carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 4 turns of an α-helix, thelength is equal to approximately 20 carbon-carbon bonds to 28carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 5 turns of an α-helix, thelength is equal to approximately 26 carbon-carbon bonds to 34carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelinkage contains approximately 4 atoms to 12 atoms, approximately 6atoms to 10 atoms, or approximately 8 atoms. Where themacrocycle-forming linker spans approximately 2 turns of the α-helix,the linkage contains approximately 7 atoms to 15 atoms, approximately 9atoms to 13 atoms, or approximately 11 atoms. Where themacrocycle-forming linker spans approximately 3 turns of the α-helix,the linkage contains approximately 13 atoms to 21 atoms, approximately15 atoms to 19 atoms, or approximately 17 atoms. Where themacrocycle-forming linker spans approximately 4 turns of the α-helix,the linkage contains approximately 19 atoms to 27 atoms, approximately21 atoms to 25 atoms, or approximately 23 atoms. Where themacrocycle-forming linker spans approximately 5 turns of the α-helix,the linkage contains approximately 25 atoms to 33 atoms, approximately27 atoms to 31 atoms, or approximately 29 atoms. Where themacrocycle-forming linker spans approximately 1 turn of the α-helix, theresulting macrocycle forms a ring containing approximately 17 members to25 members, approximately 19 members to 23 members, or approximately 21members. Where the macrocycle-forming linker spans approximately 2 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 29 members to 37 members, approximately 31 members to 35members, or approximately 33 members. Where the macrocycle-forminglinker spans approximately 3 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 44 members to 52members, approximately 46 members to 50 members, or approximately 48members. Where the macrocycle-forming linker spans approximately 4 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 59 members to 67 members, approximately 61 members to 65members, or approximately 63 members. Where the macrocycle-forminglinker spans approximately 5 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 74 members to 82members, approximately 76 members to 80 members, or approximately 78members.

In other embodiments, the invention provides peptidomimetic macrocyclesof Formula (IV) or (IVa):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid, and the terminal D and E independently optionally include acapping group;

B is a natural or non-natural amino acid, amino acid analog

[—NH-L₃-CO-], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

V and w are independently integers from 1-1000;

u is an integer from 1-10;

x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

In one example, L₁ and L₂, either alone or in combination, do not form atriazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 1. In otherembodiments of the invention, x+y+z is at least 2. In other embodimentsof the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Eachoccurrence of A, B, C, D or E in a macrocycle or macrocycle precursor ofthe invention is independently selected. For example, a sequencerepresented by the formula [A], when x is 3, encompasses embodimentswhere the amino acids are not identical, e.g. Gln-Asp-Ala as well asembodiments where the amino acids are identical, e.g. Gln-Gln-Gln. Thisapplies for any value of x, y, or z in the indicated ranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker -L₁-L₂- are shownbelow.

Preparation of Peptidomimetic Macrocycles

Peptidomimetic macrocycles of the invention may be prepared by any of avariety of methods known in the art. For example, any of the residuesindicated by “X” in Tables 1, 2, 3, or 4 may be substituted with aresidue capable of forming a crosslinker with a second residue in thesame molecule or a precursor of such a residue.

Various methods to effect formation of peptidomimetic macrocycles areknown in the art. For example, the preparation of peptidomimeticmacrocycles of Formula I is described in Schafineister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004);U.S. Pat. No. 7,192,713 and PCT application WO 2008/121767. Theα,α-disubstituted amino acids and amino acid precursors disclosed in thecited references may be employed in synthesis of the peptidomimeticmacrocycle precursor polypeptides. For example, the “S5-olefin aminoacid” is (S)-α-(2′-pentenyl) alanine and the “R8 olefin amino acid” is(R)-α-(2′-octenyl) alanine. Following incorporation of such amino acidsinto precursor polypeptides, the terminal olefins are reacted with ametathesis catalyst, leading to the formation of the peptidomimeticmacrocycle. In various embodiments, the following amino acids may beemployed in the synthesis of the peptidomimetic macrocycle:

In other embodiments, the peptidomimetic macrocyles of the invention areof Formula IV or IVa. Methods for the preparation of such macrocyclesare described, for example, in U.S. Pat. No. 7,202,332.

Additional methods of forming peptidomimetic macrocycles which areenvisioned as suitable to perform the present invention include thosedisclosed by Mustapa, M. Firouz Mohd et al., J. Org. Chem. (2003), 68,pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp.1403-1406; U.S. Pat. No. 5,364,851; U.S. Pat. No. 5,446,128; U.S. Pat.No. 5,824,483; U.S. Pat. No. 6,713,280; and U.S. Pat. No. 7,202,332. Insuch embodiments, aminoacid precursors are used containing an additionalsubstituent R— at the alpha position. Such aminoacids are incorporatedinto the macrocycle precursor at the desired positions, which may be atthe positions where the crosslinker is substituted or, alternatively,elsewhere in the sequence of the macrocycle precursor. Cyclization ofthe precursor is then effected according to the indicated method.

Assays

The properties of the peptidomimetic macrocycles of the invention areassayed, for example, by using the methods described below. In someembodiments, a peptidomimetic macrocycle of the invention has improvedbiological properties relative to a corresponding polypeptide lackingthe substituents described herein.

Assay to Determine α-Helicity.

In solution, the secondary structure of polypeptides with α-helicaldomains will reach a dynamic equilibrium between random coil structuresand α-helical structures, often expressed as a “percent helicity”. Thus,for example, alpha-helical domains are predominantly random coils insolution, with α-helical content usually under 25%. Peptidomimeticmacrocycles with optimized linkers, on the other hand, possess, forexample, an alpha-helicity that is at least two-fold greater than thatof a corresponding uncrosslinked polypeptide. In some embodiments,macrocycles of the invention will possess an alpha-helicity of greaterthan 50%. To assay the helicity of peptidomimetic macrocyles of theinvention, the compounds are dissolved in an aqueous solution (e.g. 50mM potassium phosphate solution at pH 7, or distilled H₂O, toconcentrations of 25-50 μM). Circular dichroism (CD) spectra areobtained on a spectropolarimeter (e.g., Jasco J-710) using standardmeasurement parameters (e.g. temperature, 20° C.; wavelength, 190-260nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10;response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helicalcontent of each peptide is calculated by dividing the mean residueellipticity (e.g. [Φ]222obs) by the reported value for a model helicaldecapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).

Assay to Determine Melting Temperature (Tm).

A peptidomimetic macrocycle of the invention comprising a secondarystructure such as an α-helix exhibits, for example, a higher meltingtemperature than a corresponding uncrosslinked polypeptide. Typicallypeptidomimetic macrocycles of the invention exhibit Tm of >60° C.representing a highly stable structure in aqueous solutions. To assaythe effect of macrocycle formation on melting temperature,peptidomimetic macrocycles or unmodified peptides are dissolved indistilled H₂O (e.g. at a final concentration of 50 μM) and the Tm isdetermined by measuring the change in ellipticity over a temperaturerange (e.g. 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710)using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1nm; temperature increase rate: 1° C./min; path length, 0.1 cm).

Protease Resistance Assay.

The amide bond of the peptide backbone is susceptible to hydrolysis byproteases, thereby rendering peptidic compounds vulnerable to rapiddegradation in vivo. Peptide helix formation, however, typically buriesthe amide backbone and therefore may shield it from proteolyticcleavage. The peptidomimetic macrocycles of the present invention may besubjected to in vitro trypsin proteolysis to assess for any change indegradation rate compared to a corresponding uncrosslinked polypeptide.For example, the peptidomimetic macrocycle and a correspondinguncrosslinked polypeptide are incubated with trypsin agarose and thereactions quenched at various time points by centrifugation andsubsequent HPLC injection to quantitate the residual substrate byultraviolet absorption at 280 nm Briefly, the peptidomimetic macrocycleand peptidomimetic precursor (5 mcg) are incubated with trypsin agarose(Pierce) (S/E ˜125) for 0, 10, 20, 90, and 180 minutes. Reactions arequenched by tabletop centrifugation at high speed; remaining substratein the isolated supernatant is quantified by HPLC-based peak detectionat 280 nm. The proteolytic reaction displays first order kinetics andthe rate constant, k, is determined from a plot of ln [S] versus time(k=−1× slope).

Ex Vivo Stability Assay.

Peptidomimetic macrocycles with optimized linkers possess, for example,an ex vivo half-life that is at least two-fold greater than that of acorresponding uncrosslinked polypeptide, and possess an ex vivohalf-life of 12 hours or more. For ex vivo serum stability studies, avariety of assays may be used. For example, a peptidomimetic macrocycleand a corresponding uncrosslinked polypeptide (2 mcg) are incubated withfresh mouse, rat and/or human serum (2 mL) at 37° C. for 0, 1, 2, 4, 8,and 24 hours. To determine the level of intact compound, the followingprocedure may be used: The samples are extracted by transferring 100 μlof sera to 2 ml centrifuge tubes followed by the addition of 10 μL of50% formic acid and 500 μL acetonitrile and centrifugation at 14,000 RPMfor 10 min at 4±2° C. The supernatants are then transferred to fresh 2ml tubes and evaporated on Turbovap under N₂<10 psi, 37° C. The samplesare reconstituted in 100 μL of 50:50 acetonitrile:water and submitted toLC-MS/MS analysis.

In Vitro Binding Assays.

To assess the binding and affinity of peptidomimetic macrocycles andpeptidomimetic precursors to acceptor proteins, a fluorescencepolarization assay (FPA) isused, for example. The FPA technique measuresthe molecular orientation and mobility using polarized light andfluorescent tracer. When excited with polarized light, fluorescenttracers (e.g., FITC) attached to molecules with high apparent molecularweights (e.g. FITC-labeled peptides bound to a large protein) emithigher levels of polarized fluorescence due to their slower rates ofrotation as compared to fluorescent tracers attached to smallermolecules (e.g. FITC-labeled peptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) areincubated with the acceptor protein (25-1000 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.Binding activity is measured, for example, by fluorescence polarizationon a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd valuesmay be determined by nonlinear regression analysis using, for example,Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). Apeptidomimetic macrocycle of the invention shows, in some instances,similar or lower Kd than a corresponding uncrosslinked polypeptide.

In Vitro Displacement Assays to Characterize Antagonists ofPeptide-Protein Interactions.

To assess the binding and affinity of compounds that antagonize theinteraction between a peptide and an acceptor protein, a fluorescencepolarization assay (FPA) utilizing a fluoresceinated peptidomimeticmacrocycle derived from a peptidomimetic precursor sequence is used, forexample. The FPA technique measures the molecular orientation andmobility using polarized light and fluorescent tracer. When excited withpolarized light, fluorescent tracers (e.g., FITC) attached to moleculeswith high apparent molecular weights (e.g. FITC-labeled peptides boundto a large protein) emit higher levels of polarized fluorescence due totheir slower rates of rotation as compared to fluorescent tracersattached to smaller molecules (e.g. FITC-labeled peptides that are freein solution). A compound that antagonizes the interaction between thefluoresceinated peptidomimetic macrocycle and an acceptor protein willbe detected in a competitive binding FPA experiment.

For example, putative antagonist compounds (1 nM to 1 mM) and afluoresceinated peptidomimetic macrocycle (25 nM) are incubated with theacceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL,pH 7.4) for 30 minutes at room temperature. Antagonist binding activityis measured, for example, by fluorescence polarization on a luminescencespectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determinedby nonlinear regression analysis using, for example, Graphpad Prismsoftware (GraphPad Software, Inc., San Diego, Calif.).

Any class of molecule, such as small organic molecules, peptides,oligonucleotides or proteins can be examined as putative antagonists inthis assay.

Assay for Protein-Ligand Binding by Affinity Selection-Mass Spectrometry

To assess the binding and affinity of test compounds for proteins, anaffinity-selection mass spectrometry assay is used, for example.Protein-ligand binding experiments are conducted according to thefollowing representative procedure outlined for a system-wide controlexperiment using 1 μM peptidomimetic macrocycle plus 5 μM hMDM2. A 1 μLDMSO aliquot of a 40 μM stock solution of peptidomimetic macrocycle isdissolved in 19 μL of PBS (Phosphate-buffered saline: 50 mM, pH 7.5Phosphate buffer containing 150 mM NaCl). The resulting solution ismixed by repeated pipetting and clarified by centrifugation at 10 000 gfor 10 min. To a 4 μL aliquot of the resulting supernatant is added 4 μLat of 10 μM hMDM2 in PBS. Each 8.0 μL at experimental sample thuscontains 40 pmol (1.5 μg) of protein at 5.0 μM concentration in PBS plus1 μM peptidomimetic macrocycle and 2.5% DMSO. Duplicate samples thusprepared for each concentration point are incubated for 60 min at roomtemperature, and then chilled to 4° C. prior to size-exclusionchromatography-LC-MS analysis of 5.0 μL injections. Samples containing atarget protein, protein-ligand complexes, and unbound compounds areinjected onto an SEC column, where the complexes are separated fromnon-binding component by a rapid SEC step. The SEC column eluate ismonitored using UV detectors to confirm that the early-eluting proteinfraction, which elutes in the void volume of the SEC column, is wellresolved from unbound components that are retained on the column. Afterthe peak containing the protein and protein-ligand complexes elutes fromthe primary UV detector, it enters a sample loop where it is excisedfrom the flow stream of the SEC stage and transferred directly to theLC-MS via a valving mechanism. The (M+3H)³⁺ ion of the peptidomimeticmacrocycle is observed by ESI-MS at the expected m/z, confirming thedetection of the protein-ligand complex.

Assay for Protein-Ligand Kd Titration Experiments.

To assess the binding and affinity of test compounds for proteins, aprotein-ligand Kd titration experiment is performed, for example.Protein-ligand K_(d) titrations experiments are conducted as follows: 2μL DMSO aliquots of a serially diluted stock solution of titrantpeptidomimetic macrocycle (5, 2.5, . . . , 0.098 mM) are prepared thendissolved in 38 μL of PBS. The resulting solutions are mixed by repeatedpipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0μL aliquots of the resulting supernatants is added 4.0 μL of 10 μM hMDM2in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg)of protein at 5.0 μM concentration in PBS, varying concentrations (125,62.5, . . . , 0.24 μM) of the titrant peptide, and 2.5% DMSO. Duplicatesamples thus prepared for each concentration point are incubated at roomtemperature for 30 min, then chilled to 4° C. prior to SEC-LC-MSanalysis of 2.0 μL injections. The (M+H)¹⁺, (M+2H)²⁺, (M+3H)³⁺, and/or(M+Na)¹⁺ ion is observed by ESI-MS; extracted ion chromatograms arequantified, then fit to equations to derive the binding affinity K_(d)as described in “A General Technique to Rank Protein-Ligand BindingAffinities and Determine Allosteric vs. Direct Binding Site Competitionin Compound Mixtures.” Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M.P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in “ALIS:An Affinity Selection-Mass Spectrometry System for the Discovery andCharacterization of Protein-Ligand Interactions” D. A. Annis, C.-C.Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry.Edited by Wanner K, Höfner G: Wiley-VCH; 2007:121-184. Mannhold R,Kubinyi H, Folkers G (Series Editors): Methods and Principles inMedicinal Chemistry.

Assay for Competitive Binding Experiments by Affinity Selection-MassSpectrometry

To determine the ability of test compounds to bind competitively toproteins, an affinity selection mass spectrometry assay is performed,for example. A mixture of ligands at 40 μM per component is prepared bycombining 2 μL aliquots of 400 μM stocks of each of the three compoundswith 14 μL of DMSO. Then, 1 μL at aliquots of this 40 μM per componentmixture are combined with 1 μL at DMSO aliquots of a serially dilutedstock solution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . ,0.078 mM). These 2 μL samples are dissolved in 38 μL of PBS. Theresulting solutions were mixed by repeated pipetting and clarified bycentrifugation at 10 000 g for 10 min. To 4.0 μL aliquots of theresulting supernatants is added 4.0 μL of 10 μM hMDM2 protein in PBS.Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg) ofprotein at 5.0 μM concentration in PBS plus 0.5 μM ligand, 2.5% DMSO,and varying concentrations (125, 62.5, . . . , 0.98 μM) of the titrantpeptidomimetic macrocycle. Duplicate samples thus prepared for eachconcentration point are incubated at room temperature for 60 min, thenchilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections.Additional details on these and other methods are provided in “A GeneralTechnique to Rank Protein-Ligand Binding Affinities and DetermineAllosteric vs. Direct Binding Site Competition in Compound Mixtures.”Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am.Chem. Soc. 2004, 126, 15495-15503; also in “ALIS: An AffinitySelection-Mass Spectrometry System for the Discovery andCharacterization of Protein-Ligand Interactions” D. A. Annis, C.-C.Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry.Edited by Wanner K, Hofner G: Wiley-VCH; 2007:121-184. Mannhold R,Kubinyi H, Folkers G (Series Editors): Methods and Principles inMedicinal Chemistry.

Binding Assays in Intact Cells.

It is possible to measure binding of peptides or peptidomimeticmacrocycles to their natural acceptors in intact cells byimmunoprecipitation experiments. For example, intact cells are incubatedwith fluoresceinated (FITC-labeled) compounds for 4 hrs in the absenceof serum, followed by serum replacement and further incubation thatranges from 4-18 hrs. Cells are then pelleted and incubated in lysisbuffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and proteaseinhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at14,000 rpm for 15 minutes and supernatants collected and incubated with10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed byfurther 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of50% bead slurry). After quick centrifugation, the pellets are washed inlysis buffer containing increasing salt concentration (e.g., 150, 300,500 mM). The beads are then re-equilibrated at 150 mM NaCl beforeaddition of SDS-containing sample buffer and boiling. Aftercentrifugation, the supernatants are optionally electrophoresed using4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-Pmembranes. After blocking, blots are optionally incubated with anantibody that detects FITC and also with one or more antibodies thatdetect proteins that bind to the peptidomimetic macrocycle.

Cellular Penetrability Assays.

A peptidomimetic macrocycle is, for example, more cell penetrablecompared to a corresponding uncrosslinked macrocycle. Peptidomimeticmacrocycles with optimized linkers possess, for example, cellpenetrability that is at least two-fold greater than a correspondinguncrosslinked macrocycle, and often 20% or more of the appliedpeptidomimetic macrocycle will be observed to have penetrated the cellafter 4 hours. To measure the cell penetrability of peptidomimeticmacrocycles and corresponding uncrosslinked macrocycle, intact cells areincubated with fluoresceinated peptidomimetic macrocycles orcorresponding uncrosslinked macrocycle (10 μM) for 4 hrs in serum freemedia at 37° C., washed twice with media and incubated with trypsin(0.25%) for 10 min at 37° C. The cells are washed again and resuspendedin PBS. Cellular fluorescence is analyzed, for example, by using eithera FACSCalibur flow cytometer or Cellomics' KineticScan® HCS Reader.

Cellular Efficacy Assays.

The efficacy of certain peptidomimetic macrocycles is determined, forexample, in cell-based killing assays using a variety of tumorigenic andnon-tumorigenic cell lines and primary cells derived from human or mousecell populations. Cell viability is monitored, for example, over 24-96hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μM) toidentify those that kill at EC50<10 μM. Several standard assays thatmeasure cell viability are commercially available and are optionallyused to assess the efficacy of the peptidomimetic macrocycles. Inaddition, assays that measure Annexin V and caspase activation areoptionally used to assess whether the peptidomimetic macrocycles killcells by activating the apoptotic machinery. For example, the CellTiter-glo assay is used which determines cell viability as a function ofintracellular ATP concentration.

In Vivo Stability Assay.

To investigate the in vivo stability of the peptidomimetic macrocycles,the compounds are, for example, administered to mice and/or rats by IV,IP, PO or inhalation routes at concentrations ranging from 0.1 to 50mg/kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8hrs and 24 hours post-injection. Levels of intact compound in 25 μL offresh serum are then measured by LC-MS/MS as above.

In Vivo Efficacy in Animal Models.

To determine the anti-oncogenic activity of peptidomimetic macrocyclesof the invention in vivo, the compounds are, for example, given alone(IP, IV, PO, by inhalation or nasal routes) or in combination withsub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide,doxorubicin, etoposide). In one example, 5×10⁶ RS4;11 cells (establishedfrom the bone marrow of a patient with acute lymphoblastic leukemia)that stably express luciferase are injected by tail vein in NOD-SCIDmice 3 hrs after they have been subjected to total body irradiation. Ifleft untreated, this form of leukemia is fatal in 3 weeks in this model.The leukemia is readily monitored, for example, by injecting the micewith D-luciferin (60 mg/kg) and imaging the anesthetized animals (e.g.,Xenogen In Vivo Imaging System, Caliper Life Sciences, Hopkinton,Mass.). Total body bioluminescence is quantified by integration ofphotonic flux (photons/sec) by Living Image Software (Caliper LifeSciences, Hopkinton, Mass.). Peptidomimetic macrocycles alone or incombination with sub-optimal doses of relevant chemotherapeutics agentsare, for example, administered to leukemic mice (10 days afterinjection/day 1 of experiment, in bioluminescence range of 14-16) bytail vein or IP routes at doses ranging from 0.1 mg/kg to 50 mg/kg for 7to 21 days. Optionally, the mice are imaged throughout the experimentevery other day and survival monitored daily for the duration of theexperiment. Expired mice are optionally subjected to necropsy at the endof the experiment. Another animal model is implantation into NOD-SCIDmice of DoHH2, a cell line derived from human follicular lymphoma, thatstably expresses luciferase. These in vivo tests optionally generatepreliminary pharmacokinetic, pharmacodynamic and toxicology data.

Clinical Trials.

To determine the suitability of the peptidomimetic macrocycles of theinvention for treatment of humans, clinical trials are performed. Forexample, patients diagnosed with cancer and in need of treatment areselected and separated in treatment and one or more control groups,wherein the treatment group is administered a peptidomimetic macrocycleof the invention, while the control groups receive a placebo or a knownanti-cancer drug. The treatment safety and efficacy of thepeptidomimetic macrocycles of the invention can thus be evaluated byperforming comparisons of the patient groups with respect to factorssuch as survival and quality-of-life. In this example, the patient grouptreated with a peptidomimetic macrocyle show improved long-term survivalcompared to a patient control group treated with a placebo.

Pharmaceutical Compositions and Routes of Administration

The peptidomimetic macrocycles of the invention also includepharmaceutically acceptable derivatives or prodrugs thereof A“pharmaceutically acceptable derivative” means any pharmaceuticallyacceptable salt, ester, salt of an ester, pro-drug or other derivativeof a compound of this invention which, upon administration to arecipient, is capable of providing (directly or indirectly) a compoundof this invention. Particularly favored pharmaceutically acceptablederivatives are those that increase the bioavailability of the compoundsof the invention when administered to a mammal (e.g., by increasingabsorption into the blood of an orally administered compound) or whichincreases delivery of the active compound to a biological compartment(e.g., the brain or lymphatic system) relative to the parent species.Some pharmaceutically acceptable derivatives include a chemical groupwhich increases aqueous solubility or active transport across thegastrointestinal mucosa.

In some embodiments, the peptidomimetic macrocycles of the invention aremodified by covalently or non-covalently joining appropriate functionalgroups to enhance selective biological properties. Such modificationsinclude those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism, and alter rate ofexcretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate,heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers include eithersolid or liquid carriers. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. A solid carrier can be one or more substances, which also actsas diluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

Suitable solid excipients are carbohydrate or protein fillers include,but are not limited to sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; and gums including arabic and tragacanth;as well as proteins such as gelatin and collagen. If desired,disintegrating or solubilizing agents are added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

When the compositions of this invention comprise a combination of apeptidomimetic macrocycle and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 1 to 100%, and morepreferably between about 5 to 95% of the dosage normally administered ina monotherapy regimen. In some embodiments, the additional agents areadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents are part of asingle dosage form, mixed together with the compounds of this inventionin a single composition.

Methods of Use

In one aspect, the present invention provides novel peptidomimeticmacrocycles that are useful in competitive binding assays to identifyagents which bind to the natural ligand(s) of the proteins or peptidesupon which the peptidomimetic macrocycles are modeled. For example, inthe p53/HDMX system, labeled peptidomimetic macrocycles based on p53 canbe used in a HDMX binding assay along with small molecules thatcompetitively bind to HDMX. Competitive binding studies allow for rapidin vitro evaluation and determination of drug candidates specific forthe p53/HDMX system. Such binding studies may be performed with any ofthe peptidomimetic macrocycles disclosed herein and their bindingpartners.

The invention further provides for the generation of antibodies againstthe peptidomimetic macrocycles. In some embodiments, these antibodiesspecifically bind both the peptidomimetic macrocycle and the precursorpeptides, such as p53, to which the peptidomimetic macrocycles arerelated. Such antibodies, for example, disrupt the nativeprotein-protein interaction, for example, binding between p53 and HDMX.

In other aspects, the present invention provides for both prophylacticand therapeutic methods of treating a subject at risk of (or susceptibleto) a disorder or having a disorder associated with aberrant (e.g.,insufficient or excessive) expression or activity of the moleculesincluding p53, HDM2 or HDMX.

In another embodiment, a disorder is caused, at least in part, by anabnormal level of p53 or HDM2 or HDMX, (e.g., over or under expression),or by the presence of p53 or HDM2 or HDMX exhibiting abnormal activity.As such, the reduction in the level and/or activity of p53 or HDM2 orHDMX, or the enhancement of the level and/or activity of p53 or HDM2 orHDMX, by peptidomimetic macrocycles derived from p53, is used, forexample, to ameliorate or reduce the adverse symptoms of the disorder.

In another aspect, the present invention provides methods for treatingor preventing a disease including hyperproliferative disease andinflammatory disorder by interfering with the interaction or bindingbetween binding partners, for example, between p53 and HDM2 or p53 andHDMX. These methods comprise administering an effective amount of acompound of the invention to a warm blooded animal, including a human.In some embodiments, the administration of the compounds of the presentinvention induces cell growth arrest or apoptosis.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

In some embodiments, the peptidomimetics macrocycles of the invention isused to treat, prevent, and/or diagnose cancers and neoplasticconditions. As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. A metastatic tumor can arise from a multitude of primarytumor types, including but not limited to those of breast, lung, liver,colon and ovarian origin. “Pathologic hyperproliferative” cells occur indisease states characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair. Examples of cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma, ormetastatic disorders. In some embodiments, the peptidomimeticsmacrocycles are novel therapeutic agents for controlling breast cancer,ovarian cancer, colon cancer, lung cancer, metastasis of such cancersand the like.

Examples of cancers or neoplastic conditions include, but are notlimited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer,esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer,prostate cancer, uterine cancer, cancer of the head and neck, skincancer, brain cancer, squamous cell carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, small cell lung carcinoma, non-small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma.

Examples of proliferative disorders include hematopoietic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit. Rev.Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe ovary include, but are not limited to, ovarian tumors such as,tumors of coelomic epithelium, serous tumors, mucinous tumors,endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma,Brenner tumor, surface epithelial tumors; germ cell tumors such asmature (benign) teratomas, monodermal teratomas, immature malignantteratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sexcord-stomal tumors such as, granulosa-theca cell tumors,thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma;and metastatic tumors such as Krukenberg tumors.

In other or further embodiments, the peptidomimetics macrocyclesdescribed herein are used to treat, prevent or diagnose conditionscharacterized by overactive cell death or cellular death due tophysiologic insult, etc. Some examples of conditions characterized bypremature or unwanted cell death are or alternatively unwanted orexcessive cellular proliferation include, but are not limited tohypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, and myelodysplasia.

In other or further embodiments, the peptidomimetics macrocycles of theinvention that act to decrease apoptosis are used to treat disordersassociated with an undesirable level of cell death. Thus, in someembodiments, the anti-apoptotic peptidomimetics macrocycles of theinvention are used to treat disorders such as those that lead to celldeath associated with viral infection, e.g., infection associated withinfection with human immunodeficiency virus (HIV). A wide variety ofneurological diseases are characterized by the gradual loss of specificsets of neurons. One example is Alzheimer's disease (AD). Alzheimer'sdisease is characterized by loss of neurons and synapses in the cerebralcortex and certain subcortical regions. This loss results in grossatrophy of the affected regions. Both amyloid plaques andneurofibrillary tangles are visible in brains of those afflicted by ADAlzheimer's disease has been identified as a protein misfolding disease,due to the accumulation of abnormally folded A-beta and tau proteins inthe brain. Plaques are made up of β-amyloid. β-amyloid is a fragmentfrom a larger protein called amyloid precursor protein (APP). APP iscritical to neuron growth, survival and post-injury repair. In AD, anunknown process causes APP to be cleaved into smaller fragments byenzymes through proteolysis. One of these fragments is fibrils ofβ-amyloid, which form clumps that deposit outside neurons in denseformations known as senile plaques. Plaques continue to grow intoinsoluble twisted fibers within the nerve cell, often called tangles.Disruption of the interaction between β-amyloid and its native receptoris therefore important in the treatment of AD. The anti-apoptoticpeptidomimetics macrocycles of the invention are used, in someembodiments, in the treatment of AD and other neurological disordersassociated with cell apoptosis. Such neurological disorders includeAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis(ALS) retinitis pigmentosa, spinal muscular atrophy, and various formsof cerebellar degeneration. The cell loss in these diseases does notinduce an inflammatory response, and apoptosis appears to be themechanism of cell death.

In addition, a number of hematologic diseases are associated with adecreased production of blood cells. These disorders include anemiaassociated with chronic disease, aplastic anemia, chronic neutropenia,and the myelodysplastic syndromes. Disorders of blood cell production,such as myelodysplastic syndrome and some forms of aplastic anemia, areassociated with increased apoptotic cell death within the bone marrow.These disorders could result from the activation of genes that promoteapoptosis, acquired deficiencies in stromal cells or hematopoieticsurvival factors, or the direct effects of toxins and mediators ofimmune responses. Two common disorders associated with cell death aremyocardial infarctions and stroke. In both disorders, cells within thecentral area of ischemia, which is produced in the event of acute lossof blood flow, appear to die rapidly as a result of necrosis. However,outside the central ischemic zone, cells die over a more protracted timeperiod and morphologically appear to die by apoptosis. In other orfurther embodiments, the anti-apoptotic peptidomimetics macrocycles ofthe invention are used to treat all such disorders associated withundesirable cell death.

Some examples of neurologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary CerebralHemorrhage Amyloidosis, Reactive Amyloidosis, Familial AmyloidNephropathy with Urticaria and Deafness, Muckle-Wells Syndrome,Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, FamilialAmyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, IsolatedCardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid,Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis,Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease,Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis,a prion-mediated disease, and Huntington's Disease.

In another embodiment, the peptidomimetics macrocycles described hereinare used to treat, prevent or diagnose inflammatory disorders. Numeroustypes of inflammatory disorders exist. Certain inflammatory diseases areassociated with the immune system, for example, autoimmune diseases.Autoimmune diseases arise from an overactive immune response of the bodyagainst substances and tissues normally present in the body, i.e. selfantigens. In other words, the immune system attacks its own cells.Autoimmune diseases are a major cause of immune-mediated diseases.Rheumatoid arthritis is an example of an autoimmune disease, in whichthe immune system attacks the joints, where it causes inflammation (i.e.arthritis) and destruction. It can also damage some organs, such as thelungs and skin. Rheumatoid arthritis can lead to substantial loss offunctioning and mobility. Rheumatoid arthritis is diagnosed with bloodtests especially the rheumatoid factor test. Some examples of autoimmunediseases that are treated with the peptidomimetics macrocycles describedherein include, but are not limited to, acute disseminatedencephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis,antiphospholipid antibody syndrome (APS), autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune inner ear disease, Bechet's disease,bullous pemphigoid, coeliac disease, Chagas disease, Churg-Strausssyndrome, chronic obstructive pulmonary disease (COPD), Crohn's disease,dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture'ssyndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto'sdisease, Hidradenitis suppurativa, idiopathic thrombocytopenic purpura,inflammatory bowl disease (IBD), interstitial cystitis, lupuserythematosus, morphea, multiple sclerosis, myasthenia gravis,narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anaemia,Polymyositis, polymyalgia rheumatica, primary biliary cirrhosis,psoriasis, rheumatoid arthritis, schizophrenia, scleroderma, Sjögren'ssyndrome, temporal arteritis (also known as “giant cell arteritis”),Takayasu's arteritis, Vasculitis, Vitiligo, and Wegener'sgranulomatosis.

Some examples of other types of inflammatory disorders that are treatedwith the peptidomimetics macrocycles described herein include, but arenot limited to, allergy including allergic rhinitis/sinusitis, skinallergies (urticaria/hives, angioedema, atopic dermatitis), foodallergies, drug allergies, insect allergies, and rare allergic disorderssuch as mastocytosis, asthma, arthritis including osteoarthritis,rheumatoid arthritis, and spondyloarthropathies, primary angitis of theCNS, sarcoidosis, organ transplant rejection, fibromyalgia, fibrosis,pancreatitis, and pelvic inflammatory disease.

Examples of cardiovascular disorders (e.g., inflammatory disorders) thatare treated or prevented with the peptidomimetics macrocycles of theinvention include, but are not limited to, aortic valve stenosis,atherosclerosis, myocardial infarction, stroke, thrombosis, aneurism,heart failure, ischemic heart disease, angina pectoris, sudden cardiacdeath, hypertensive heart disease; non-coronary vessel disease, such asarteriolosclerosis, small vessel disease, nephropathy,hypertriglyceridemia, hypercholesterolemia, hyperlipidemia,xanthomatosis, asthma, hypertension, emphysema and chronic pulmonarydisease; or a cardiovascular condition associated with interventionalprocedures (“procedural vascular trauma”), such as restenosis followingangioplasty, placement of a shunt, stent, synthetic or natural excisiongrafts, indwelling catheter, valve or other implantable devices.Preferred cardiovascular disorders include atherosclerosis, myocardialinfarction, aneurism, and stroke.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1 Synthesis of 6-chlorotryptophan Fmoc Amino Acids

Tert-butyl 6-chloro-3-formyl-1H-indole-1-carboxylate, 1. To a stirredsolution of dry DMF (12 mL) was added dropwise POCl₃ (3.92 mL, 43 mmol,1.3 equiv) at 0° C. under Argon. The solution was stirred at the sametemperature for 20 min before a solution of 6-chloroindole (5.0 g, 33mmol, 1 eq.) in dry DMF (30 mL) was added dropwise. The resultingmixture was allowed to warm to room temperature and stirred for anadditional 2.5 h. Water (50 mL) was added and the solution wasneutralized with 4M aqueous NaOH (pH ˜8). The resulting solid wasfiltered off, washed with water and dried under vacuum. This materialwas directly used in the next step without additional purification. To astirred solution of the crude formyl indole (33 mmol, 1 eq.) in THF (150mL) was added successively Boc₂O (7.91 g, 36.3 mmol, 1.1 equiv) and DMAP(0.4 g, 3.3 mmol, 0.1 equiv) at room temperature under N₂. The resultingmixture was stirred at room temperature for 1.5 h and the solvent wasevaporated under reduced pressure. The residue was taken up in EtOAc andwashed with 1N HCl, dried and concentrated to give the formyl indole 1(9 g, 98% over 2 steps) as a white solid. ¹H NMR (CDCl₃) δ: 1.70 (s,Boc, 9H); 7.35 (dd, 1H); 8.21 (m, 3H); 10.07 (s, 1H).

Tert-butyl 6-chloro-3-(hydroxymethyl)-1H-indole-1-carboxylate, 2. To asolution of compound 1 (8.86 g, 32 mmol, 1 eq.) in ethanol (150 mL) wasadded NaBH₄ (2.4 g, 63 mmol, 2 eq.). The reaction was stirred for 3 h atroom temperature. The reaction mixture was concentrated and the residuewas poured into diethyl ether and water. The organic layer wasseparated, dried over magnesium sulfate and concentrated to give a whitesolid (8.7 g, 98%). This material was directly used in the next stepwithout additional purification. ¹H NMR (CDCl₃) δ: 1.65 (s, Boc, 9H);4.80 (s, 2H, CH₂); 7.21 (dd, 1H); 7.53 (m, 2H); 8.16 (bs, 1H).

Tert-butyl 3-(bromomethyl)-6-chloro-1H-indole-1-carboxylate, 3. To asolution of compound 2 (4.1 g, 14.6 mmol, 1 eq.) in dichloromethane (50mL) under argon was added a solution of triphenylphosphine (4.59 g, 17.5mmol, 1.2 eq.) in dichloromethane (50 mL) at −40° C. The reactionsolution was stirred an additional 30 min at 40° C. Then NBS (3.38 g, 19mmol, 1.3 eq.) was added. The resulting mixture was allowed to warm toroom temperature and stirred overnight. Dichloromethane was evaporated,Carbon Tetrachloride (100 mL) was added and the mixture was stirred for1 h and filtrated. The filtrate was concentrated, loaded in a silicaplug and quickly eluted with 25% EtOAc in Hexanes. The solution wasconcentrated to give a white foam (3.84 g, 77%). ¹H NMR (CDCl₃) δ: 1.66(s, Boc, 9H); 4.63 (s, 2H, CH₂); 7.28 (dd, 1H); 7.57 (d, 1H); 7.64 (bs,1H); 8.18 (bs, 1H).

αMe-6Cl-Trp(Boc)-Ni—S—BPB, 4. To S-Ala-Ni—S—BPB (2.66 g, 5.2 mmol, 1eq.) and KO-tBu (0.87 g, 7.8 mmol, 1.5 eq.) was added 50 mL of DMF underargon. The bromide derivative compound 3 (2.68 g, 7.8 mmol, 1.5 eq.) insolution of DMF (5.0 mL) was added via syringe. The reaction mixture wasstirred at ambient temperature for 1 h. The solution was then quenchedwith 5% aqueous acetic acid and diluted with water. The desired productwas extracted in dichloromethane, dried and concentrated. The oilyproduct 4 was purified by flash chromatography (solid loading) on normalphase using EtOAc and Hexanes as eluents to give a red solid (1.78 g,45% yield). αMe-6C1-Trp(Boc)-Ni—S—BPB, 4: M+H calc. 775.21, M+H obs.775.26; ¹H NMR (CDCl₃) δ: 1.23 (s, 3H, αMe); 1.56 (m, 11H, Boc+CH₂);1.82-2.20 (m, 4H, 2CH₂); 3.03 (m, 1H, CH_(a)); 3.24 (m, 2H, CH₂); 3.57and 4.29 (AB system, 2H, CH₂ (benzyl), J=12.8 Hz); 6.62 (d, 2H); 6.98(d, 1H); 7.14 (m, 2H); 7.23 (m, 1H); 7.32-7.36 (m, 5H); 7.50 (m, 2H);7.67 (bs, 1H); 7.98 (d, 2H); 8.27 (m, 2H).

Fmoc-αMe-6Cl-Trp(Boc)-OH, 6. To a solution of 3N HCl/MeOH (1/3, 15 mL)at 50° C. was added a solution of compound 4 (1.75 g, 2.3 mmol, 1 eq.)in MeOH (5 ml) dropwise. The starting material disappeared within 3-4 h.The acidic solution was then cooled to 0° C. with an ice bath andquenched with an aqueous solution of Na₂CO₃ (1.21 g, 11.5 mmol, 5 eq.).Methanol was removed and 8 more equivalents of Na₂CO₃ (1.95 g, 18.4mmol) were added to the suspension. The Nickel scavenging EDTA disodiumsalt dihydrate (1.68 g, 4.5 mmol, 2 eq.) was then added and thesuspension was stirred for 2 h. A solution of Fmoc-OSu (0.84 g, 2.5mmol, 1.1 eq.) in acetone (50 mL) was added and the reaction was stirredovernight. Afterwards, the reaction was diluted with diethyl ether and1N HCl. The organic layer was then dried over magnesium sulfate andconcentrated in vacuo. The desired product 6 was purified on normalphase using acetone and dichloromethane as eluents to give a white foam(0.9 g, 70% yield). Fmoc-αMe-6Cl-Trp(Boc)-OH, 6: M+H calc. 575.19, M+Hobs. 575.37; ¹H NMR (CDCl₃) 1.59 (s, 9H, Boc); 1.68 (s, 3H, Me); 3.48(bs, 2H, CH₂); 4.22 (m, 1H, CH); 4.39 (bs, 2H, CH₂); 5.47 (s, 1H, NH);7.10 (m, 1H); 7.18 (m, 2H); 7.27 (m, 2H); 7.39 (m, 2H); 7.50 (m, 2H);7.75 (d, 2H); 8.12 (bs, 1H).

6Cl-Trp(Boc)-Ni—S—BPB, 5. To Gly-Ni—S—BPB (4.6 g, 9.2 mmol, 1 eq.) andKO-tBu (1.14 g, 10.1 mmol, 1.1 eq.) was added 95 mL of DMF under argon.The bromide derivative compound 3 (3.5 g, 4.6 mmol, 1.1 eq.) in solutionof DMF (10 mL) was added via syringe. The reaction mixture was stirredat ambient temperature for 1 h. The solution was then quenched with 5%aqueous acetic acid and diluted with water. The desired product wasextracted in dichloromethane, dried and concentrated. The oily product 5was purified by flash chromatography (solid loading) on normal phaseusing EtOAc and Hexanes as eluents to give a red solid (5 g, 71% yield).6Cl-Trp(Boc)-Ni—S—BPB, 5: M+H calc. 761.20, M+H obs. 761.34; ¹H NMR(CDCl₃) δ: 1.58 (m, 11H, Boc+CH₂); 1.84 (m, 1H); 1.96 (m, 1H); 2.24 (m,2H, CH₂); 3.00 (m, 1H, CH_(a)); 3.22 (m, 2H, CH₂); 3.45 and 4.25 (ABsystem, 2H, CH₂ (benzyl), J=12.8 Hz); 4.27 (m, 1H, CH_(a)); 6.65 (d,2H); 6.88 (d, 1H); 7.07 (m, 2H); 7.14 (m, 2H); 7.28 (m, 3H); 7.35-7.39(m, 2H); 7.52 (m, 2H); 7.96 (d, 2H); 8.28 (m, 2H).

Fmoc-6Cl-Trp(Boc)-OH, 7. To a solution of 3N HCl/MeOH (1/3, 44 mL) at50° C. was added a solution of compound 5 (5 g, 6.6 mmol, 1 eq.) in MeOH(10 ml) dropwise. The starting material disappeared within 3-4 h. Theacidic solution was then cooled to 0° C. with an ice bath and quenchedwith an aqueous solution of Na₂CO₃ (3.48 g, 33 mmol, 5 eq.). Methanolwas removed and 8 more equivalents of Na₂CO₃ (5.57 g, 52 mmol) wereadded to the suspension. The Nickel scavenging EDTA disodium saltdihydrate (4.89 g, 13.1 mmol, 2 eq.) and the suspension was stirred for2 h. A solution of Fmoc-OSu (2.21 g, 6.55 mmol, 1.1 eq.) in acetone (100mL) was added and the reaction was stirred overnight. Afterwards, thereaction was diluted with diethyl ether and 1N HCl. The organic layerwas then dried over magnesium sulfate and concentrated in vacuo. Thedesired product 7 was purified on normal phase using acetone anddichloromethane as eluents to give a white foam (2.6 g, 69% yield).Fmoc-6Cl-Trp(Boc)-OH, 7: M+H calc. 561.17, M+H obs. 561.37; ¹H NMR(CDCl₃) 1.63 (s, 9H, Boc); 3.26 (m, 2H, CH₂); 4.19 (m, 1H, CH); 4.39 (m,2H, CH₂); 4.76 (m, 1H); 5.35 (d, 1H, NH); 7.18 (m, 2H); 7.28 (m, 2H);7.39 (m, 3H); 7.50 (m, 2H); 7.75 (d, 2H); 8.14 (bs, 1H).

Example 2 Peptidomimetic Macrocycles of the Invention

Peptidomimetic macrocycles were synthesized, purified and analyzed aspreviously described and as described below (Schafmeister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); andU.S. Pat. No. 7,192,713). Peptidomimetic macrocycles were designed byreplacing two or more naturally occurring amino acids with thecorresponding synthetic amino acids. Substitutions were made at i andi+4, and i and i+7 positions. Peptide synthesis was performed eithermanually or on an automated peptide synthesizer (Applied Biosystems,model 433A), using solid phase conditions, rink amide AM resin(Novabiochem), and Fmoc main-chain protecting group chemistry. For thecoupling of natural Fmoc-protected amino acids (Novabiochem), 10equivalents of amino acid and a 1:1:2 molar ratio of coupling reagentsHBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural amino acids (4equiv) were coupled with a 1:1:2 molar ratio of HATU (AppliedBiosystems)/HOBt/DIEA. The N-termini of the synthetic peptides wereacetylated, while the C-termini were amidated.

Purification of cross-linked compounds was achieved by high performanceliquid chromatography (HPLC) (Varian ProStar) on a reverse phase C18column (Varian) to yield the pure compounds. Chemical composition of thepure products was confirmed by LC/MS mass spectrometry (Micromass LCTinterfaced with Agilent 1100 HPLC system) and amino acid analysis(Applied Biosystems, model 420A).

Table 4 shows a list of peptidomimetic macrocycles of the inventionprepared.

TABLE 4 Observed Exact mass SP Seq Mass M + 2 (m/e) SP-1Ac-LSQETF$r8DLWKLL$EN-NH2 2068.13 1035.07 1035.36 SP-2Ac-LSQETF$r8NLWKLL$QN-NH2 2066.16 1034.08 1034.31 SP-3Ac-LSQQTF$r8NLWRLL$QN-NH2 2093.18 1047.59 1047.73 SP-4Ac-QSQQTF$r8NLWKLL$QN-NH2 2080.15 1041.08 1041.31 SP-5Ac-QSQQTF$r8NLWRLL$QN-NH2 2108.15 1055.08 1055.32 SP-6Ac-QSQQTA$r8NLWRLL$QN-NH2 2032.12 1017.06 1017.24 SP-7Ac-QAibQQTF$r8NLWRLL$QN-NH2 2106.17 1054.09 1054.34 SP-8Ac-QSQQTFSNLWRLLPQN-NH2 2000.02 1001.01 1001.26 SP-9Ac-QSQQTF$/r8NLWRLL$/QN-NH2 2136.18 1069.09 1069.37 SP-10Ac-QSQAibTF$r8NLWRLL$QN-NH2 2065.15 1033.58 1033.71 SP-11Ac-QSQQTF$r8NLWRLL$AN-NH2 2051.13 1026.57 1026.70 SP-12Ac-ASQQTF$r8NLWRLL$QN-NH2 2051.13 1026.57 1026.90 SP-13Ac-QSQQTF$r8ALWRLL$QN-NH2 2065.15 1033.58 1033.41 SP-14Ac-QSQETF$r8NLWRLL$QN-NH2 2109.14 1055.57 1055.70 SP-15Ac-RSQQTF$r8NLWRLL$QN-NH2 2136.20 1069.10 1069.17 SP-16Ac-RSQQTF$r8NLWRLL$EN-NH2 2137.18 1069.59 1069.75 SP-17Ac-LSQETFSDLWKLLPEN-NH2 1959.99 981.00 981.24 SP-18Ac-QSQ$TFS$LWRLLPQN-NH2 2008.09 1005.05 1004.97 SP-19Ac-QSQQ$FSN$WRLLPQN-NH2 2036.06 1019.03 1018.86 SP-20Ac-QSQQT$SNL$RLLPQN-NH2 1917.04 959.52 959.32 SP-21Ac-QSQQTF$NLW$LLPQN-NH2 2007.06 1004.53 1004.97 SP-22Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2 2310.26 1156.13 1156.52 SP-23Ac-QSQQTF$r8NLWRLL$RN-NH2 2136.20 1069.10 1068.94 SP-24Ac-QSQRTF$r8NLWRLL$QN-NH2 2136.20 1069.10 1068.94 SP-25Ac-QSQQTF$r8NNleWRLL$QN-NH2 2108.15 1055.08 1055.44 SP-26Ac-QSQQTF$r8NLWRNleL$QN-NH2 2108.15 1055.08 1055.84 SP-27Ac-QSQQTF$r8NLWRLNle$QN-NH2 2108.15 1055.08 1055.12 SP-28Ac-QSQQTY$r8NLWRLL$QN-NH2 2124.15 1063.08 1062.92 SP-29Ac-RAibQQTF$r8NLWRLL$QN-NH2 2134.22 1068.11 1068.65 SP-30Ac-MPRFMDYWEGLN-NH2 1598.70 800.35 800.45 SP-31Ac-RSQQRF$r8NLWRLL$QN-NH2 2191.25 1096.63 1096.83 SP-32Ac-QSQQRF$r8NLWRLL$QN-NH2 2163.21 1082.61 1082.87 SP-33Ac-RAibQQRF$r8NLWRLL$QN-NH2 2189.27 1095.64 1096.37 SP-34Ac-RSQQRF$r8NFWRLL$QN-NH2 2225.23 1113.62 1114.37 SP-35Ac-RSQQRF$r8NYWRLL$QN-NH2 2241.23 1121.62 1122.37 SP-36Ac-RSQQTF$r8NLWQLL$QN-NH2 2108.15 1055.08 1055.29 SP-37Ac-QSQQTF$r8NLWQAmlL$QN-NH2 2094.13 1048.07 1048.32 SP-38Ac-QSQQTF$r8NAmlWRLL$QN-NH2 2122.17 1062.09 1062.35 SP-39Ac-NlePRF$r8DYWEGL$QN-NH2 1869.98 935.99 936.20 SP-40Ac-NlePRF$r8NYWRLL$QN-NH2 1952.12 977.06 977.35 SP-41Ac-RF$r8NLWRLL$Q-NH2 1577.96 789.98 790.18 SP-42Ac-QSQQTF$r8N2ffWRLL$QN-NH2 2160.13 1081.07 1081.40 SP-43Ac-QSQQTF$r8N3ffWRLL$QN-NH2 2160.13 1081.07 1081.34 SP-44Ac-QSQQTF#r8NLWRLL#QN-NH2 2080.12 1041.06 1041.34 SP-45Ac-RSQQTA$r8NLWRLL$QN-NH2 2060.16 1031.08 1031.38 SP-46Ac-QSQQTF%r8NLWRLL%QN-NH2 2110.17 1056.09 1056.55 SP-47HepQSQ$TFSNLWRLLPQN-NH2 2051.10 1026.55 1026.82 SP-48HepQSQ$TF$r8NLWRLL$QN-NH2 2159.23 1080.62 1080.89 SP-49Ac-QSQQTF$r8NL6cIWRLL$QN-NH2 2142.11 1072.06 1072.35 SP-50Ac-QSQQTF$r8NLMe6cIwRLL$QN-NH2 2156.13 1079.07 1079.27 SP-51Ac-LTFEHYWAQLTS-NH2 1535.74 768.87 768.91 SP-52 Ac-LTF$HYW$QLTS-NH21585.83 793.92 794.17 SP-53 Ac-LTFE$YWA$LTS-NH2 1520.79 761.40 761.67SP-54 Ac-LTF$zr8HYWAQL$zS-NH2 1597.87 799.94 800.06 SP-55Ac-LTF$r8HYWRQL$S-NH2 1682.93 842.47 842.72 SP-56Ac-QS$QTFStNLWRLL$s8QN-NH2 2145.21 1073.61 1073.90 SP-57Ac-QSQQTASNLWRLLPQN-NH2 1923.99 963.00 963.26 SP-58Ac-QSQQTA$/r8NLWRLL$/QN-NH2 2060.15 1031.08 1031.24 SP-59Ac-ASQQTF$/r8NLWRLL$/QN-NH2 2079.16 1040.58 1040.89 SP-60Ac-$SQQ$FSNLWRLLAibQN-NH2 2009.09 1005.55 1005.86 SP-61Ac-QS$QTF$NLWRLLAibQN-NH2 2023.10 1012.55 1012.79 SP-62Ac-QSQQ$FSN$WRLLAibQN-NH2 2024.06 1013.03 1013.31 SP-63Ac-QSQQTF$NLW$LLAibQN-NH2 1995.06 998.53 998.87 SP-64Ac-QSQQTFS$LWR$LAibQN-NH2 2011.06 1006.53 1006.83 SP-65Ac-QSQQTFSNLW$LLA$N-NH2 1940.02 971.01 971.29 SP-66Ac-$/SQQ$/FSNLWRLLAibQN-NH2 2037.12 1019.56 1019.78 SP-67Ac-QS$/QTF$/NLWRLLAibQN-NH2 2051.13 1026.57 1026.90 SP-68Ac-QSQQ$/FSN$/WRLLAibQN-NH2 2052.09 1027.05 1027.36 SP-69Ac-QSQQTF$/NLW$/LLAibQN-NH2 2023.09 1012.55 1013.82 SP-70Ac-QSQ$TFS$LWRLLAibQN-NH2 1996.09 999.05 999.39 SP-71Ac-QSQ$/TFS$/LWRLLAibQN-NH2 2024.12 1013.06 1013.37 SP-72Ac-QS$/QTFSt//NLWRLL$/s8QN-NH2 2201.27 1101.64 1102.00 SP-73Ac-$r8SQQTFS$LWRLLAibQN-NH2 2038.14 1020.07 1020.23 SP-74Ac-QSQ$r8TFSNLW$LLAibQN-NH2 1996.08 999.04 999.32 SP-75Ac-QSQQTFS$r8LWRLLA$N-NH2 2024.12 1013.06 1013.37 SP-76Ac-QS$r5QTFStNLW$LLAibQN-NH2 2032.12 1017.06 1017.39 SP-77Ac-$/r8SQQTFS$/LWRLLAibQN-NH2 2066.17 1034.09 1034.80 SP-78Ac-QSQ$/r8TFSNLW$/LLAibQN-NH2 2024.11 1013.06 1014.34 SP-79Ac-QSQQTFS$/r8LWRLLA$/N-NH2 2052.15 1027.08 1027.16 SP-80Ac-QS$/r5QTFSt//NLW$/LLAibQN-NH2 2088.18 1045.09 1047.10 SP-81Ac-QSQQTFSNLWRLLAibQN-NH2 1988.02 995.01 995.31 SP-82Hep/QSQ$/TF$/r8NLWRLL$/QN-NH2 2215.29 1108.65 1108.93 SP-83Ac-ASQQTF$r8NLRWLL$QN-NH2 2051.13 1026.57 1026.90 SP-84Ac-QSQQTF$/r8NLWRLL$/Q-NH2 2022.14 1012.07 1012.66 SP-85Ac-QSQQTF$r8NLWRLL$Q-NH2 1994.11 998.06 998.42 SP-86Ac-AAARAA$r8AAARAA$AA-NH2 1515.90 758.95 759.21 SP-87Ac-LTFEHYWAQLTSA-NH2 1606.78 804.39 804.59 SP-88 Ac-LTF$r8HYWAQL$SA-NH21668.90 835.45 835.67 SP-89 Ac-ASQQTFSNLWRLLPQN-NH2 1943.00 972.50973.27 SP-90 Ac-QS$QTFStNLW$r5LLAibQN-NH2 2032.12 1017.06 1017.30 SP-91Ac-QSQQTFAibNLWRLLAibQN-NH2 1986.04 994.02 994.19 SP-92Ac-QSQQTFNleNLWRLLNleQN-NH2 2042.11 1022.06 1022.23 SP-93Ac-QSQQTF$/r8NLWRLLAibQN-NH2 2082.14 1042.07 1042.23 SP-94Ac-QSQQTF$/r8NLWRLLNleQN-NH2 2110.17 1056.09 1056.29 SP-95Ac-QSQQTFAibNLWRLL$/QN-NH2 2040.09 1021.05 1021.25 SP-96Ac-QSQQTFNleNLWRLL$/QN-NH2 2068.12 1035.06 1035.31 SP-97Ac-QSQQTF%r8NL6cIWRNleL%QN-NH2 2144.13 1073.07 1073.32 SP-98Ac-QSQQTF%r8NLMe6cIWRLL%QN-NH2 2158.15 1080.08 1080.31 SP-101Ac-FNle$YWE$L-NH2 1160.63 — 1161.70 SP-102 Ac-F$r8AYWELL$A-NH2 1344.75 —1345.90 SP-103 Ac-F$r8AYWQLL$A-NH2 1343.76 — 1344.83 SP-104Ac-NlePRF$r8NYWELL$QN-NH2 1925.06 963.53 963.69 SP-105Ac-NlePRF$r8DYWRLL$QN-NH2 1953.10 977.55 977.68 SP-106Ac-NlePRF$r8NYWRLL$Q-NH2 1838.07 920.04 920.18 SP-107Ac-NlePRF$r8NYWRLL$-NH2 1710.01 856.01 856.13 SP-108Ac-QSQQTF$r8DLWRLL$QN-NH2 2109.14 1055.57 1055.64 SP-109Ac-QSQQTF$r8NLWRLL$EN-NH2 2109.14 1055.57 1055.70 SP-110Ac-QSQQTF$r8NLWRLL$QD-NH2 2109.14 1055.57 1055.64 SP-111Ac-QSQQTF$r8NLWRLL$S-NH2 1953.08 977.54 977.60 SP-112Ac-ESQQTF$r8NLWRLL$QN-NH2 2109.14 1055.57 1055.70 SP-113Ac-LTF$r8NLWRNleL$Q-NH2 1635.99 819.00 819.10 SP-114Ac-LRF$r8NLWRNleL$Q-NH2 1691.04 846.52 846.68 SP-115Ac-QSQQTF$r8NWWRNleL$QN-NH2 2181.15 1091.58 1091.64 SP-116Ac-QSQQTF$r8NLWRNleL$Q-NH2 1994.11 998.06 998.07 SP-117Ac-QTF$r8NLWRNleL$QN-NH2 1765.00 883.50 883.59 SP-118Ac-NlePRF$r8NWWRLL$QN-NH2 1975.13 988.57 988.75 SP-119Ac-NlePRF$r8NWWRLL$A-NH2 1804.07 903.04 903.08 SP-120Ac-TSFAEYWNLLSP-NH2 1467.70 734.85 734.90 SP-121 Ac-QTF$r8HWWSQL$S-NH21651.85 826.93 827.12 SP-122 Ac-FM$YWE$L-NH2 1178.58 — 1179.64 SP-123Ac-QTFEHWWSQLLS-NH2 1601.76 801.88 801.94 SP-124Ac-QSQQTF$r8NLAmwRLNle$QN-NH2 2122.17 1062.09 1062.24 SP-125Ac-FMAibY6cIWEAc3cL-NH2 1130.47 — 1131.53 SP-126 Ac-FNle$Y6cIWE$L-NH21194.59 — 1195.64 SP-127 Ac-F$zr8AY6cIWEAc3cL$z-NH2 1277.63 639.821278.71 SP-128 Ac-F$r8AY6cIWEAc3cL$A-NH2 1348.66 — 1350.72 SP-129Ac-NlePRF$r8NY6cIWRLL$QN-NH2 1986.08 994.04 994.64 SP-130Ac-AF$r8AAWALA$A-NH2 1223.71 — 1224.71 SP-131 Ac-TF$r8AAWRLA$Q-NH21395.80 698.90 399.04 SP-132 Pr-TF$r8AAWRLA$Q-NH2 1409.82 705.91 706.04SP-133 Ac-QSQQTF%r8NLWRNleL%QN-NH2 2110.17 1056.09 1056.22 SP-134Ac-LTF%r8HYWAQL%SA-NH2 1670.92 836.46 836.58 SP-135Ac-NlePRF%r8NYWRLL%QN-NH2 1954.13 978.07 978.19 SP-136Ac-NlePRF%r8NY6cIWRLL%QN-NH2 1988.09 995.05 995.68 SP-137Ac-LTF%r8HY6cIWAQL%S-NH2 1633.84 817.92 817.93 SP-138Ac-QS%QTF%StNLWRLL%s8QN-NH2 2149.24 1075.62 1075.65 SP-139Ac-LTF%r8HY6cIWRQL%S-NH2 1718.91 860.46 860.54 SP-140Ac-QSQQTF%r8NL6cIWRLL%QN-NH2 2144.13 1073.07 1073.64 SP-141Ac-%r8SQQTFS%LWRLLAibQN-NH2 2040.15 1021.08 1021.13 SP-142Ac-LTF%r8HYWAQL%S-NH2 1599.88 800.94 801.09 SP-143 Ac-TSF%r8QYWNLL%P-NH21602.88 802.44 802.58 SP-147 Ac-LTFEHYWAQLTS-NH2 1535.74 768.87 769.5SP-152 Ac-F$er8AY6cIWEAc3cL$e-NH2 1277.63 639.82 1278.71 SP-153Ac-AF$r8AAWALA$A-NH2 1277.63 639.82 1277.84 SP-154 Ac-TF$r8AAWRLA$Q-NH21395.80 698.90 699.04 SP-155 Pr-TF$r8AAWRLA$Q-NH2 1409.82 705.91 706.04SP-156 Ac-LTF$er8HYWAQL$eS-NH2 1597.87 799.94 800.44 SP-159Ac-CCPGCCBaQSQQTF$r8NLWRLL$QN-NH2 2745.30 1373.65 1372.99 SP-160Ac-CCPGCCBaQSQQTA$r8NLWRLL$QN- 2669.27 1335.64 1336.09 NH2 SP-161Ac-CCPGCCBaNlePRF$r8NYWRLL$QN-NH2 2589.26 1295.63 1296.2 SP-162Ac-LTF$/r8HYWAQL$/S-NH2 1625.90 813.95 814.18 SP-163Ac-F%r8HY6cIWRAc3cL%-NH2 1372.72 687.36 687.59 SP-164Ac-QTF%r8HWWSQL%S-NH2 1653.87 827.94 827.94 SP-165 Ac-LTA$r8HYWRQL$S-NH21606.90 804.45 804.66 SP-166 Ac-Q$r8QQTFSN$WRLLAibQN-NH2 2080.12 1041.061041.61 SP-167 Ac-QSQQ$r8FSNLWR$LAibQN-NH2 2066.11 1034.06 1034.58SP-168 Ac-F$r8AYWEAc3cL$A-NH2 1314.70 658.35 1315.88 SP-169Ac-F$r8AYWEAc3cL$S-NH2 1330.70 666.35 1331.87 SP-170Ac-F$r8AYWEAc3cL$Q-NH2 1371.72 686.86 1372.72 SP-171Ac-F$r8AYWEAibL$S-NH2 1332.71 667.36 1334.83 SP-172 Ac-F$r8AYWEAL$S-NH21318.70 660.35 1319.73 SP-173 Ac-F$r8AYWEQL$S-NH2 1375.72 688.86 1377.53SP-174 Ac-F$r8HYWEQL$S-NH2 1441.74 721.87 1443.48 SP-175Ac-F$r8HYWAQL$S-NH2 1383.73 692.87 1385.38 SP-176 Ac-F$r8HYWAAc3cL$S-NH21338.71 670.36 1340.82 SP-177 Ac-F$r8HYWRAc3cL$S-NH2 1423.78 712.89713.04 SP-178 Ac-F$r8AYWEAc3cL#A-NH2 1300.69 651.35 1302.78 SP-179Ac-NlePTF%r8NYWRLL%QN-NH2 1899.08 950.54 950.56 SP-180Ac-TF$r8AAWRAL$Q-NH2 1395.80 698.90 699.13 SP-181 Ac-TSF%r8HYWAQL%S-NH21573.83 787.92 787.98 SP-184 Ac-F%r8AY6cIWEAc3cL%A-NH2 1350.68 676.34676.91 SP-185 Ac-LTF$r8HYWAQI$S-NH2 1597.87 799.94 800.07 SP-186Ac-LTF$r8HYWAQNle$S-NH2 1597.87 799.94 800.07 SP-187Ac-LTF$r8HYWAQL$A-NH2 1581.87 791.94 792.45 SP-188Ac-LTF$r8HYWAQL$Abu-NH2 1595.89 798.95 799.03 SP-189Ac-LTF$r8HYWAbuQL$S-NH2 1611.88 806.94 807.47 SP-190Ac-LTF$er8AYWAQL$eS-NH2 1531.84 766.92 766.96 SP-191Ac-LAF$r8HYWAQL$S-NH2 1567.86 784.93 785.49 SP-192 Ac-LAF$r8AYWAQL$S-NH21501.83 751.92 752.01 SP-193 Ac-LTF$er8AYWAQL$eA-NH2 1515.85 758.93758.97 SP-194 Ac-LAF$r8AYWAQL$A-NH2 1485.84 743.92 744.05 SP-195Ac-LTF$r8NLWANleL$Q-NH2 1550.92 776.46 776.61 SP-196Ac-LTF$r8NLWANleL$A-NH2 1493.90 747.95 1495.6 SP-197Ac-LTF$r8ALWANleL$Q-NH2 1507.92 754.96 755 SP-198Ac-LAF$r8NLWANleL$Q-NH2 1520.91 761.46 761.96 SP-199Ac-LAF$r8ALWANleL$A-NH2 1420.89 711.45 1421.74 SP-200Ac-A$r8AYWEAc3cL$A-NH2 1238.67 620.34 1239.65 SP-201Ac-F$r8AYWEAc3cL$AA-NH2 1385.74 693.87 1386.64 SP-202Ac-F$r8AYWEAc3cL$Abu-NH2 1328.72 665.36 1330.17 SP-203Ac-F$r8AYWEAc3cL$Nle-NH2 1356.75 679.38 1358.22 SP-204Ac-F$r5AYWEAc3cL$s8A-NH2 1314.70 658.35 1315.51 SP-205Ac-F$AYWEAc3cL$r8A-NH2 1314.70 658.35 1315.66 SP-206Ac-F$r8AYWEAc3cI$A-NH2 1314.70 658.35 1316.18 SP-207Ac-F$r8AYWEAc3cNle$A-NH2 1314.70 658.35 1315.66 SP-208Ac-F$r8AYWEAmlL$A-NH2 1358.76 680.38 1360.21 SP-209Ac-F$r8AYWENleL$A-NH2 1344.75 673.38 1345.71 SP-210Ac-F$r8AYWQAc3cL$A-NH2 1313.72 657.86 1314.7 SP-211Ac-F$r8AYWAAc3cL$A-NH2 1256.70 629.35 1257.56 SP-212Ac-F$r8AYWAbuAc3cL$A-NH2 1270.71 636.36 1272.14 SP-213Ac-F$r8AYWNleAc3cL$A-NH2 1298.74 650.37 1299.67 SP-214Ac-F$r8AbuYWEAc3cL$A-NH2 1328.72 665.36 1329.65 SP-215Ac-F$r8NleYWEAc3cL$A-NH2 1356.75 679.38 1358.66 SP-2165-FAM-BaLTFEHYWAQLTS-NH2 1922.82 962.41 962.87 SP-2175-FAM-BaLTF%r8HYWAQL%S-NH2 1986.96 994.48 994.97 SP-218Ac-LTF$r8HYWAQhL$S-NH2 1611.88 806.94 807 SP-219 Ac-LTF$r8HYWAQTle$S-NH21597.87 799.94 799.97 SP-220 Ac-LTF$r8HYWAQAdm$S-NH2 1675.91 838.96839.09 SP-221 Ac-LTF$r8HYWAQhCha$S-NH2 1651.91 826.96 826.98 SP-222Ac-LTF$r8HYWAQCha$S-NH2 1637.90 819.95 820.02 SP-223Ac-LTF$r8HYWAc6cQL$S-NH2 1651.91 826.96 826.98 SP-224Ac-LTF$r8HYWAc5cQL$S-NH2 1637.90 819.95 820.02 SP-225Ac-LThF$r8HYWAQL$S-NH2 1611.88 806.94 807 SP-226 Ac-LTIgl$r8HYWAQL$S-NH21625.90 813.95 812.99 SP-227 Ac-LTF$r8HYWAQChg$S-NH2 1623.88 812.94812.99 SP-228 Ac-LTF$r8HYWAQF$S-NH2 1631.85 816.93 816.99 SP-229Ac-LTF$r8HYWAQIgl$S-NH2 1659.88 830.94 829.94 SP-230Ac-LTF$r8HYWAQCba$S-NH2 1609.87 805.94 805.96 SP-231Ac-LTF$r8HYWAQCpg$S-NH2 1609.87 805.94 805.96 SP-232Ac-LTF$r8HhYWAQL$S-NH2 1611.88 806.94 807 SP-233 Ac-F$r8AYWEAc3chL$A-NH21328.72 665.36 665.43 SP-234 Ac-F$r8AYWEAc3cTle$A-NH2 1314.70 658.351315.62 SP-235 Ac-F$r8AYWEAc3cAdm$A-NH2 1392.75 697.38 697.47 SP-236Ac-F$r8AYWEAc3chCha$A-NH2 1368.75 685.38 685.34 SP-237Ac-F$r8AYWEAc3cCha$A-NH2 1354.73 678.37 678.38 SP-238Ac-F$r8AYWEAc6cL$A-NH2 1356.75 679.38 679.42 SP-239Ac-F$r8AYWEAc5cL$A-NH2 1342.73 672.37 672.46 SP-240Ac-hF$r8AYWEAc3cL$A-NH2 1328.72 665.36 665.43 SP-241Ac-Igl$r8AYWEAc3cL$A-NH2 1342.73 672.37 671.5 SP-243Ac-F$r8AYWEAc3cF$A-NH2 1348.69 675.35 675.35 SP-244Ac-F$r8AYWEAc3cIgl$A-NH2 1376.72 689.36 688.37 SP-245Ac-F$r8AYWEAc3cCba$A-NH2 1326.70 664.35 664.47 SP-246Ac-F$r8AYWEAc3cCpg$A-NH2 1326.70 664.35 664.39 SP-247Ac-F$r8AhYWEAc3cL$A-NH2 1328.72 665.36 665.43 SP-248Ac-F$r8AYWEAc3cL$Q-NH2 1371.72 686.86 1372.87 SP-249Ac-F$r8AYWEAibL$A-NH2 1316.72 659.36 1318.18 SP-250 Ac-F$r8AYWEAL$A-NH21302.70 652.35 1303.75 SP-251 Ac-LAF$r8AYWAAL$A-NH2 1428.82 715.41715.49 SP-252 Ac-LTF$r8HYWAAc3cL$S-NH2 1552.84 777.42 777.5 SP-253Ac-NleTF$r8HYWAQL$S-NH2 1597.87 799.94 800.04 SP-254Ac-VTF$r8HYWAQL$S-NH2 1583.85 792.93 793.04 SP-255 Ac-FTF$r8HYWAQL$S-NH21631.85 816.93 817.02 SP-256 Ac-WTF$r8HYWAQL$S-NH2 1670.86 836.43 836.85SP-257 Ac-RTF$r8HYWAQL$S-NH2 1640.88 821.44 821.9 SP-258Ac-KTF$r8HYWAQL$S-NH2 1612.88 807.44 807.91 SP-259Ac-LNleF$r8HYWAQL$S-NH2 1609.90 805.95 806.43 SP-260Ac-LVF$r8HYWAQL$S-NH2 1595.89 798.95 798.93 SP-261 Ac-LFF$r8HYWAQL$S-NH21643.89 822.95 823.38 SP-262 Ac-LWF$r8HYWAQL$S-NH2 1682.90 842.45 842.55SP-263 Ac-LRF$r8HYWAQL$S-NH2 1652.92 827.46 827.52 SP-264Ac-LKF$r8HYWAQL$S-NH2 1624.91 813.46 813.51 SP-265Ac-LTF$r8NleYWAQL$S-NH2 1573.89 787.95 788.05 SP-266Ac-LTF$r8VYWAQL$S-NH2 1559.88 780.94 780.98 SP-267 Ac-LTF$r8FYWAQL$S-NH21607.88 804.94 805.32 SP-268 Ac-LTF$r8WYWAQL$S-NH2 1646.89 824.45 824.86SP-269 Ac-LTF$r8RYWAQL$S-NH2 1616.91 809.46 809.51 SP-270Ac-LTF$r8KYWAQL$S-NH2 1588.90 795.45 795.48 SP-271Ac-LTF$r8HNleWAQL$S-NH2 1547.89 774.95 774.98 SP-272Ac-LTF$r8HVWAQL$S-NH2 1533.87 767.94 767.95 SP-273 Ac-LTF$r8HFWAQL$S-NH21581.87 791.94 792.3 SP-274 Ac-LTF$r8HWWAQL$S-NH2 1620.88 811.44 811.54SP-275 Ac-LTF$r8HRWAQL$S-NH2 1590.90 796.45 796.52 SP-276Ac-LTF$r8HKWAQL$S-NH2 1562.90 782.45 782.53 SP-277Ac-LTF$r8HYWNleQL$S-NH2 1639.91 820.96 820.98 SP-278Ac-LTF$r8HYWVQL$S-NH2 1625.90 813.95 814.03 SP-279 Ac-LTF$r8HYWFQL$S-NH21673.90 837.95 838.03 SP-280 Ac-LTF$r8HYWWQL$S-NH2 1712.91 857.46 857.5SP-281 Ac-LTF$r8HYWKQL$S-NH2 1654.92 828.46 828.49 SP-282Ac-LTF$r8HYWANleL$S-NH2 1582.89 792.45 792.52 SP-283Ac-LTF$r8HYWAVL$S-NH2 1568.88 785.44 785.49 SP-284 Ac-LTF$r8HYWAFL$S-NH21616.88 809.44 809.47 SP-285 Ac-LTF$r8HYWAWL$S-NH2 1655.89 828.95 829SP-286 Ac-LTF$r8HYWARL$S-NH2 1625.91 813.96 813.98 SP-287Ac-LTF$r8HYWAQL$Nle-NH2 1623.92 812.96 813.39 SP-288Ac-LTF$r8HYWAQL$V-NH2 1609.90 805.95 805.99 SP-289 Ac-LTF$r8HYWAQL$F-NH21657.90 829.95 830.26 SP-290 Ac-LTF$r8HYWAQL$W-NH2 1696.91 849.46 849.5SP-291 Ac-LTF$r8HYWAQL$R-NH2 1666.94 834.47 834.56 SP-292Ac-LTF$r8HYWAQL$K-NH2 1638.93 820.47 820.49 SP-293Ac-Q$r8QQTFSN$WRLLAibQN-NH2 2080.12 1041.06 1041.54 SP-294Ac-QSQQ$r8FSNLWR$LAibQN-NH2 2066.11 1034.06 1034.58 SP-295Ac-LT2Pal$r8HYWAQL$S-NH2 1598.86 800.43 800.49 SP-296Ac-LT3Pal$r8HYWAQL$S-NH2 1598.86 800.43 800.49 SP-297Ac-LT4Pal$r8HYWAQL$S-NH2 1598.86 800.43 800.49 SP-298Ac-LTF2CF3$r8HYWAQL$S-NH2 1665.85 833.93 834.01 SP-299Ac-LTF2CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 SP-300Ac-LTF2Me$r8HYWAQL$S-NH2 1611.88 806.94 807 SP-301Ac-LTF3CI$r8HYWAQL$S-NH2 1631.83 816.92 816.99 SP-302Ac-LTF4CF3$r8HYWAQL$S-NH2 1665.85 833.93 833.94 SP-303Ac-LTF4tBu$r8HYWAQL$S-NH2 1653.93 827.97 828.02 SP-304Ac-LTF5F$r8HYWAQL$S-NH2 1687.82 844.91 844.96 SP-305Ac-LTF$r8HY3BthAAQL$S-NH2 1614.83 808.42 808.48 SP-306Ac-LTF2Br$r8HYWAQL$S-NH2 1675.78 838.89 838.97 SP-307Ac-LTF4Br$r8HYWAQL$S-NH2 1675.78 838.89 839.86 SP-308Ac-LTF2CI$r8HYWAQL$S-NH2 1631.83 816.92 816.99 SP-309Ac-LTF4CI$r8HYWAQL$S-NH2 1631.83 816.92 817.36 SP-310Ac-LTF3CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 SP-311Ac-LTF4CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 SP-312Ac-LTF34CI2$r8HYWAQL$S-NH2 1665.79 833.90 833.94 SP-313Ac-LTF34F2$r8HYWAQL$S-NH2 1633.85 817.93 817.95 SP-314Ac-LTF35F2$r8HYWAQL$S-NH2 1633.85 817.93 817.95 SP-315Ac-LTDip$r8HYWAQL$S-NH2 1673.90 837.95 838.01 SP-316Ac-LTF2F$r8HYWAQL$S-NH2 1615.86 808.93 809 SP-317Ac-LTF3F$r8HYWAQL$S-NH2 1615.86 808.93 809 SP-318Ac-LTF4F$r8HYWAQL$S-NH2 1615.86 808.93 809 SP-319Ac-LTF4I$r8HYWAQL$S-NH2 1723.76 862.88 862.94 SP-320Ac-LTF3Me$r8HYWAQL$S-NH2 1611.88 806.94 807.07 SP-321Ac-LTF4Me$r8HYWAQL$S-NH2 1611.88 806.94 807 SP-322Ac-LT1Nal$r8HYWAQL$S-NH2 1647.88 824.94 824.98 SP-323Ac-LT2Nal$r8HYWAQL$S-NH2 1647.88 824.94 825.06 SP-324Ac-LTF3CF3$r8HYWAQL$S-NH2 1665.85 833.93 834.01 SP-325Ac-LTF4NO2$r8HYWAQL$S-NH2 1642.85 822.43 822.46 SP-326Ac-LTF3NO2$r8HYWAQL$S-NH2 1642.85 822.43 822.46 SP-327Ac-LTF$r82ThiYWAQL$S-NH2 1613.83 807.92 807.96 SP-328Ac-LTF$r8HBipWAQL$S-NH2 1657.90 829.95 830.01 SP-329Ac-LTF$r8HF4tBuWAQL$S-NH2 1637.93 819.97 820.02 SP-330Ac-LTF$r8HF4CF3WAQL$S-NH2 1649.86 825.93 826.02 SP-331Ac-LTF$r8HF4CIWAQL$S-NH2 1615.83 808.92 809.37 SP-332Ac-LTF$r8HF4MeWAQL$S-NH2 1595.89 798.95 799.01 SP-333Ac-LTF$r8HF4BrWAQL$S-NH2 1659.78 830.89 830.98 SP-334Ac-LTF$r8HF4CNWAQL$S-NH2 1606.87 804.44 804.56 SP-335Ac-LTF$r8HF4NO2WAQL$S-NH2 1626.86 814.43 814.55 SP-336Ac-LTF$r8H1NalWAQL$S-NH2 1631.89 816.95 817.06 SP-337Ac-LTF$r8H2NalWAQL$S-NH2 1631.89 816.95 816.99 SP-338Ac-LTF$r8HWAQL$S-NH2 1434.80 718.40 718.49 SP-339Ac-LTF$r8HY1NalAQL$S-NH2 1608.87 805.44 805.52 SP-340Ac-LTF$r8HY2NalAQL$S-NH2 1608.87 805.44 805.52 SP-341Ac-LTF$r8HYWAQI$S-NH2 1597.87 799.94 800.07 SP-342Ac-LTF$r8HYWAQNle$S-NH2 1597.87 799.94 800.44 SP-343Ac-LTF$er8HYWAQL$eA-NH2 1581.87 791.94 791.98 SP-344Ac-LTF$r8HYWAQL$Abu-NH2 1595.89 798.95 799.03 SP-345Ac-LTF$r8HYWAbuQL$S-NH2 1611.88 806.94 804.47 SP-346Ac-LAF$r8HYWAQL$S-NH2 1567.86 784.93 785.49 SP-347Ac-LTF$r8NLWANleL$Q-NH2 1550.92 776.46 777.5 SP-348Ac-LTF$r8ALWANleL$Q-NH2 1507.92 754.96 755.52 SP-349Ac-LAF$r8NLWANleL$Q-NH2 1520.91 761.46 762.48 SP-350Ac-F$r8AYWAAc3CI$A-NH2 1256.70 629.35 1257.56 SP-351Ac-LTF$r8AYWAAL$S-NH2 1474.82 738.41 738.55 SP-352 Ac-LVF$r8AYWAQL$S-NH21529.87 765.94 766 SP-353 Ac-LTF$r8AYWAbuQL$S-NH2 1545.86 773.93 773.92SP-354 Ac-LTF$r8AYWNleQL$S-NH2 1573.89 787.95 788.17 SP-355Ac-LTF$r8AbuYWAQL$S-NH2 1545.86 773.93 773.99 SP-356Ac-LTF$r8AYWHQL$S-NH2 1597.87 799.94 799.97 SP-357 Ac-LTF$r8AYWKQL$S-NH21588.90 795.45 795.53 SP-358 Ac-LTF$r8AYWOQL$S-NH2 1574.89 788.45 788.5SP-359 Ac-LTF$r8AYWRQL$S-NH2 1616.91 809.46 809.51 SP-360Ac-LTF$r8AYWSQL$S-NH2 1547.84 774.92 774.96 SP-361 Ac-LTF$r8AYWRAL$S-NH21559.89 780.95 780.95 SP-362 Ac-LTF$r8AYWRQL$A-NH2 1600.91 801.46 801.52SP-363 Ac-LTF$r8AYWRAL$A-NH2 1543.89 772.95 773.03 SP-364Ac-LTF$r5HYWAQL$s8S-NH2 1597.87 799.94 799.97 SP-365Ac-LTF$HYWAQL$r8S-NH2 1597.87 799.94 799.97 SP-366 Ac-LTF$r8HYWAAL$S-NH21540.84 771.42 771.48 SP-367 Ac-LTF$r8HYWAAbuL$S-NH2 1554.86 778.43778.51 SP-368 Ac-LTF$r8HYWALL$S-NH2 1582.89 792.45 792.49 SP-369Ac-F$r8AYWHAL$A-NH2 1310.72 656.36 656.4 SP-370 Ac-F$r8AYWAAL$A-NH21244.70 623.35 1245.61 SP-371 Ac-F$r8AYWSAL$A-NH2 1260.69 631.35 1261.6SP-372 Ac-F$r8AYWRAL$A-NH2 1329.76 665.88 1330.72 SP-373Ac-F$r8AYWKAL$A-NH2 1301.75 651.88 1302.67 SP-374 Ac-F$r8AYWOAL$A-NH21287.74 644.87 1289.13 SP-375 Ac-F$r8VYWEAc3cL$A-NH2 1342.73 672.371343.67 SP-376 Ac-F$r8FYWEAc3cL$A-NH2 1390.73 696.37 1392.14 SP-377Ac-F$r8WYWEAc3cL$A-NH2 1429.74 715.87 1431.44 SP-378Ac-F$r8RYWEAc3cL$A-NH2 1399.77 700.89 700.95 SP-379Ac-F$r8KYWEAc3cL$A-NH2 1371.76 686.88 686.97 SP-380Ac-F$r8ANleWEAc3cL$A-NH2 1264.72 633.36 1265.59 SP-381Ac-F$r8AVWEAc3cL$A-NH2 1250.71 626.36 1252.2 SP-382Ac-F$r8AFWEAc3cL$A-NH2 1298.71 650.36 1299.64 SP-383Ac-F$r8AWWEAc3cL$A-NH2 1337.72 669.86 1338.64 SP-384Ac-F$r8ARWEAc3cL$A-NH2 1307.74 654.87 655 SP-385 Ac-F$r8AKWEAc3cL$A-NH21279.73 640.87 641.01 SP-386 Ac-F$r8AYWVAc3cL$A-NH2 1284.73 643.37643.38 SP-387 Ac-F$r8AYWFAc3cL$A-NH2 1332.73 667.37 667.43 SP-388Ac-F$r8AYWWAc3cL$A-NH2 1371.74 686.87 686.97 SP-389Ac-F$r8AYWRAc3cL$A-NH2 1341.76 671.88 671.94 SP-390Ac-F$r8AYWKAc3cL$A-NH2 1313.75 657.88 657.88 SP-391 Ac-F$r8AYWEVLSA-NH21330.73 666.37 666.47 SP-392 Ac-F$r8AYWEFL$A-NH2 1378.73 690.37 690.44SP-393 Ac-F$r8AYWEWL$A-NH2 1417.74 709.87 709.91 SP-394Ac-F$r8AYWERL$A-NH2 1387.77 694.89 1388.66 SP-395 Ac-F$r8AYWEKL$A-NH21359.76 680.88 1361.21 SP-396 Ac-F$r8AYWEAc3cL$V-NH2 1342.73 672.371343.59 SP-397 Ac-F$r8AYWEAc3cL$F-NH2 1390.73 696.37 1392.58 SP-398Ac-F$r8AYWEAc3cL$W-NH2 1429.74 715.87 1431.29 SP-399Ac-F$r8AYWEAc3cL$R-NH2 1399.77 700.89 700.95 SP-400Ac-F$r8AYWEAc3cL$K-NH2 1371.76 686.88 686.97 SP-401Ac-F$r8AYWEAc3cL$AV-NH2 1413.77 707.89 707.91 SP-402Ac-F$r8AYWEAc3cL$AF-NH2 1461.77 731.89 731.96 SP-403Ac-F$r8AYWEAc3cL$AW-NH2 1500.78 751.39 751.5 SP-404Ac-F$r8AYWEAc3cL$AR-NH2 1470.80 736.40 736.47 SP-405Ac-F$r8AYWEAc3cL$AK-NH2 1442.80 722.40 722.41 SP-406Ac-F$r8AYWEAc3cL$AH-NH2 1451.76 726.88 726.93 SP-407Ac-LTF2NO2$r8HYWAQL$S-NH2 1642.85 822.43 822.54 SP-408Ac-LTA$r8HYAAQL$S-NH2 1406.79 704.40 704.5 SP-409 Ac-LTF$r8HYAAQL$S-NH21482.82 742.41 742.47 SP-410 Ac-QSQQTF$r8NLWALL$AN-NH2 1966.07 984.04984.38 SP-411 Ac-QAibQQTF$r8NLWALL$AN-NH2 1964.09 983.05 983.42 SP-412Ac-QAibQQTF$r8ALWALL$AN-NH2 1921.08 961.54 961.59 SP-413Ac-AAAATF$r8AAWAAL$AA-NH2 1608.90 805.45 805.52 SP-414Ac-F$r8AAWRAL$Q-NH2 1294.76 648.38 648.48 SP-415 Ac-TF$r8AAWAAL$Q-NH21310.74 656.37 1311.62 SP-416 Ac-TF$r8AAWRAL$A-NH2 1338.78 670.39 670.46SP-417 Ac-VF$r8AAWRAL$Q-NH2 1393.82 697.91 697.99 SP-418Ac-AF$r8AAWAAL$A-NH2 1223.71 612.86 1224.67 SP-420 Ac-TF$r8AAWKAL$Q-NH21367.80 684.90 684.97 SP-421 Ac-TF$r8AAWOAL$Q-NH2 1353.78 677.89 678.01SP-422 Ac-TF$r8AAWSAL$Q-NH2 1326.73 664.37 664.47 SP-423Ac-LTF$r8AAWRAL$Q-NH2 1508.89 755.45 755.49 SP-424 Ac-F$r8AYWAQL$A-NH21301.72 651.86 651.96 SP-425 Ac-F$r8AWWAAL$A-NH2 1267.71 634.86 634.87SP-426 Ac-F$r8AWWAQL$A-NH2 1324.73 663.37 663.43 SP-427Ac-F$r8AYWEAL$-NH2 1231.66 616.83 1232.93 SP-428 Ac-F$r8AYWAAL$-NH21173.66 587.83 1175.09 SP-429 Ac-F$r8AYWKAL$-NH2 1230.72 616.36 616.44SP-430 Ac-F$r8AYWOAL$-NH2 1216.70 609.35 609.48 SP-431Ac-F$r8AYWQAL$-NH2 1230.68 616.34 616.44 SP-432 Ac-F$r8AYWAQL$-NH21230.68 616.34 616.37 SP-433 Ac-F$r8HYWDQL$S-NH2 1427.72 714.86 714.86SP-434 Ac-F$r8HFWEQL$S-NH2 1425.74 713.87 713.98 SP-435Ac-F$r8AYWHQL$S-NH2 1383.73 692.87 692.96 SP-436 Ac-F$r8AYWKQL$S-NH21374.77 688.39 688.45 SP-437 Ac-F$r8AYWOQL$S-NH2 1360.75 681.38 681.49SP-438 Ac-F$r8HYWSQL$S-NH2 1399.73 700.87 700.95 SP-439Ac-F$r8HWWEQL$S-NH2 1464.76 733.38 733.44 SP-440 Ac-F$r8HWWAQL$S-NH21406.75 704.38 704.43 SP-441 Ac-F$r8AWWHQL$S-NH2 1406.75 704.38 704.43SP-442 Ac-F$r8AWWKQL$S-NH2 1397.79 699.90 699.92 SP-443Ac-F$r8AWWOQL$S-NH2 1383.77 692.89 692.96 SP-444 Ac-F$r8HWWSQL$S-NH21422.75 712.38 712.42 SP-445 Ac-LTF$r8NYWANleL$Q-NH2 1600.90 801.45801.52 SP-446 Ac-LTF$r8NLWAQL$Q-NH2 1565.90 783.95 784.06 SP-447Ac-LTF$r8NYWANleL$A-NH2 1543.88 772.94 773.03 SP-448Ac-LTF$r8NLWAQL$A-NH2 1508.88 755.44 755.49 SP-449Ac-LTF$r8AYWANleL$Q-NH2 1557.90 779.95 780.06 SP-450Ac-LTF$r8ALWAQL$Q-NH2 1522.89 762.45 762.45 SP-451Ac-LAF$r8NYWANleL$Q-NH2 1570.89 786.45 786.5 SP-452Ac-LAF$r8NLWAQL$Q-NH2 1535.89 768.95 769.03 SP-453Ac-LAF$r8AYWANleL$A-NH2 1470.86 736.43 736.47 SP-454Ac-LAF$r8ALWAQL$A-NH2 1435.86 718.93 719.01 SP-455 Ac-LAF$r8AYWAAL$A-NH21428.82 715.41 715.41 SP-456 Ac-F$r8AYWEAc3cL$AAib-NH2 1399.75 700.88700.95 SP-457 Ac-F$r8AYWAQL$AA-NH2 1372.75 687.38 687.78 SP-458Ac-F$r8AYWAAc3cL$AA-NH2 1327.73 664.87 664.84 SP-459Ac-F$r8AYWSAc3cL$AA-NH2 1343.73 672.87 672.9 SP-460Ac-F$r8AYWEAc3cL$AS-NH2 1401.73 701.87 701.84 SP-461Ac-F$r8AYWEAc3cL$AT-NH2 1415.75 708.88 708.87 SP-462Ac-F$r8AYWEAc3cL$AL-NH2 1427.79 714.90 714.94 SP-463Ac-F$r8AYWEAc3cL$AQ-NH2 1442.76 722.38 722.41 SP-464Ac-F$r8AFWEAc3cL$AA-NH2 1369.74 685.87 685.93 SP-465Ac-F$r8AWWEAc3cL$AA-NH2 1408.75 705.38 705.39 SP-466Ac-F$r8AYWEAc3cL$SA-NH2 1401.73 701.87 701.99 SP-467Ac-F$r8AYWEAL$AA-NH2 1373.74 687.87 687.93 SP-468 Ac-F$r8AYWENleL$AA-NH21415.79 708.90 708.94 SP-469 Ac-F$r8AYWEAc3cL$AbuA-NH2 1399.75 700.88700.95 SP-470 Ac-F$r8AYWEAc3cL$NleA-NH2 1427.79 714.90 714.86 SP-471Ac-F$r8AYWEAibL$NleA-NH2 1429.80 715.90 715.97 SP-472Ac-F$r8AYWEAL$NleA-NH2 1415.79 708.90 708.94 SP-473Ac-F$r8AYWENleL$NleA-NH2 1457.83 729.92 729.96 SP-474Ac-F$r8AYWEAibL$Abu-NH2 1330.73 666.37 666.39 SP-475Ac-F$r8AYWENleL$Abu-NH2 1358.76 680.38 680.39 SP-476Ac-F$r8AYWEAL$Abu-NH2 1316.72 659.36 659.36 SP-477 Ac-LTF$r8AFWAQL$S-NH21515.85 758.93 759.12 SP-478 Ac-LTF$r8AWWAQL$S-NH2 1554.86 778.43 778.51SP-479 Ac-LTF$r8AYWAQI$S-NH2 1531.84 766.92 766.96 SP-480Ac-LTF$r8AYWAQNle$S-NH2 1531.84 766.92 766.96 SP-481Ac-LTF$r8AYWAQL$SA-NH2 1602.88 802.44 802.48 SP-482Ac-LTF$r8AWWAQL$A-NH2 1538.87 770.44 770.89 SP-483 Ac-LTF$r8AYWAQI$A-NH21515.85 758.93 759.42 SP-484 Ac-LTF$r8AYWAQNle$A-NH2 1515.85 758.93759.42 SP-485 Ac-LTF$r8AYWAQL$AA-NH2 1586.89 794.45 794.94 SP-486Ac-LTF$r8HWWAQL$S-NH2 1620.88 811.44 811.47 SP-487 Ac-LTF$r8HRWAQL$S-NH21590.90 796.45 796.52 SP-488 Ac-LTF$r8HKWAQL$S-NH2 1562.90 782.45 782.53SP-489 Ac-LTF$r8HYWAQL$W-NH2 1696.91 849.46 849.5 SP-491Ac-F$r8AYWAbuAL$A-NH2 1258.71 630.36 630.5 SP-492 Ac-F$r8AbuYWEAL$A-NH21316.72 659.36 659.51 SP-493 Ac-NlePRF%r8NYWRLL%QN-NH2 1954.13 978.07978.54 SP-494 Ac-TSF%r8HYWAQL%S-NH2 1573.83 787.92 787.98 SP-495Ac-LTF%r8AYWAQL%S-NH2 1533.86 767.93 768 SP-496 Ac-HTF$r8HYWAQL$S-NH21621.84 811.92 811.96 SP-497 Ac-LHF$r8HYWAQL$S-NH2 1633.88 817.94 818.02SP-498 Ac-LTF$r8HHWAQL$S-NH2 1571.86 786.93 786.94 SP-499Ac-LTF$r8HYWHQL$S-NH2 1663.89 832.95 832.38 SP-500 Ac-LTF$r8HYWAHL$S-NH21606.87 804.44 804.48 SP-501 Ac-LTF$r8HYWAQL$H-NH2 1647.89 824.95 824.98SP-502 Ac-LTF$r8HYWAQL$S-NHPr 1639.91 820.96 820.98 SP-503Ac-LTF$r8HYWAQL$S-NHsBu 1653.93 827.97 828.02 SP-504Ac-LTF$r8HYWAQL$S-NHiBu 1653.93 827.97 828.02 SP-505Ac-LTF$r8HYWAQL$S-NHBn 1687.91 844.96 844.44 SP-506Ac-LTF$r8HYWAQL$S-NHPe 1700.92 851.46 851.99 SP-507Ac-LTF$r8HYWAQL$S-NHChx 1679.94 840.97 841.04 SP-508Ac-ETF$r8AYWAQL$S-NH2 1547.80 774.90 774.96 SP-509 Ac-STF$r8AYWAQL$S-NH21505.79 753.90 753.94 SP-510 Ac-LEF$r8AYWAQL$S-NH2 1559.84 780.92 781.25SP-511 Ac-LSF$r8AYWAQL$S-NH2 1517.83 759.92 759.93 SP-512Ac-LTF$r8EYWAQL$S-NH2 1589.85 795.93 795.97 SP-513 Ac-LTF$r8SYWAQL$S-NH21547.84 774.92 774.96 SP-514 Ac-LTF$r8AYWEQL$S-NH2 1589.85 795.93 795.9SP-515 Ac-LTF$r8AYWAEL$S-NH2 1532.83 767.42 766.96 SP-516Ac-LTF$r8AYWASL$S-NH2 1490.82 746.41 746.46 SP-517 Ac-LTF$r8AYWAQL$E-NH21573.85 787.93 787.98 SP-518 Ac-LTF2CN$r8HYWAQL$S-NH2 1622.86 812.43812.47 SP-519 Ac-LTF3CI$r8HYWAQL$S-NH2 1631.83 816.92 816.99 SP-520Ac-LTDip$r8HYWAQL$S-NH2 1673.90 837.95 838.01 SP-521Ac-LTF$r8HYWAQTle$S-NH2 1597.87 799.94 800.04 SP-522Ac-F$r8AY6cIWEAL$A-NH2 1336.66 669.33 1338.56 SP-523Ac-F$r8AYdI6brWEAL$A-NH2 1380.61 691.31 692.2 SP-524Ac-F$r8AYdI6fWEAL$A-NH2 1320.69 661.35 1321.61 SP-525Ac-F$r8AYdI4mWEAL$A-NH2 1316.72 659.36 659.36 SP-526Ac-F$r8AYdI5cIWEAL$A-NH2 1336.66 669.33 669.35 SP-527Ac-F$r8AYdI7mWEAL$A-NH2 1316.72 659.36 659.36 SP-528Ac-LTF%r8HYWAQL%A-NH2 1583.89 792.95 793.01 SP-529Ac-LTF$r8HCouWAQL$S-NH2 1679.87 840.94 841.38 SP-530Ac-LTFEHCouWAQLTS-NH2 1617.75 809.88 809.96 SP-531Ac-LTA$r8HCouWAQL$S-NH2 1603.84 802.92 803.36 SP-532Ac-F$r8AYWEAL$AbuA-NH2 1387.75 694.88 694.88 SP-533 Ac-F$r8AYWEAI$AA-NH21373.74 687.87 687.93 SP-534 Ac-F$r8AYWEANle$AA-NH2 1373.74 687.87687.93 SP-535 Ac-F$r8AYWEAmIL$AA-NH2 1429.80 715.90 715.97 SP-536Ac-F$r8AYWQAL$AA-NH2 1372.75 687.38 687.48 SP-537 Ac-F$r8AYWAAL$AA-NH21315.73 658.87 658.92 SP-538 Ac-F$r8AYWAbuAL$AA-NH2 1329.75 665.88665.95 SP-539 Ac-F$r8AYWNleAL$AA-NH2 1357.78 679.89 679.94 SP-540Ac-F$r8AbuYWEAL$AA-NH2 1387.75 694.88 694.96 SP-541Ac-F$r8NleYWEAL$AA-NH2 1415.79 708.90 708.94 SP-542 Ac-F$r8FYWEAL$AA-NH21449.77 725.89 725.97 SP-543 Ac-LTF$r8HYWAQhL$S-NH2 1611.88 806.94 807SP-544 Ac-LTF$r8HYWAQAdm$S-NH2 1675.91 838.96 839.04 SP-545Ac-LTF$r8HYWAQIgl$S-NH2 1659.88 830.94 829.94 SP-546Ac-F$r8AYWAQL$AA-NH2 1372.75 687.38 687.48 SP-547 Ac-LTF$r8ALWAQL$Q-NH21522.89 762.45 762.52 SP-548 Ac-F$r8AYWEAL$AA-NH2 1373.74 687.87 687.93SP-549 Ac-F$r8AYWENleL$AA-NH2 1415.79 708.90 708.94 SP-550Ac-F$r8AYWEAibL$Abu-NH2 1330.73 666.37 666.39 SP-551Ac-F$r8AYWENleL$Abu-NH2 1358.76 680.38 680.38 SP-552Ac-F$r8AYWEAL$Abu-NH2 1316.72 659.36 659.36 SP-553Ac-F$r8AYWEAc3cL$AbuA-NH2 1399.75 700.88 700.95 SP-554Ac-F$r8AYWEAc3cL$NleA-NH2 1427.79 714.90 715.01 SP-555H-LTF$r8AYWAQL$S-NH2 1489.83 745.92 745.95 SP-556mdPEG3-LTF$r8AYWAQL$S-NH2 1679.92 840.96 840.97 SP-557mdPEG7-LTF$r8AYWAQL$S-NH2 1856.02 929.01 929.03 SP-558Ac-F$r8ApmpEt6cIWEAL$A-NH2 1470.71 736.36 788.17 SP-559Ac-LTF3CI$r8AYWAQL$S-NH2 1565.81 783.91 809.18 SP-560Ac-LTF3CI$r8HYWAQL$A-NH2 1615.83 808.92 875.24 SP-561Ac-LTF3CI$r8HYWWQL$S-NH2 1746.87 874.44 841.65 SP-562Ac-LTF3CI$r8AYWWQL$S-NH2 1680.85 841.43 824.63 SP-563Ac-LTF$r8AYWWQL$S-NH2 1646.89 824.45 849.98 SP-564 Ac-LTF$r8HYWWQL$A-NH21696.91 849.46 816.67 SP-565 Ac-LTF$r8AYWWQL$A-NH2 1630.89 816.45 776.15SP-566 Ac-LTF4F$r8AYWAQL$S-NH2 1549.83 775.92 776.15 SP-567Ac-LTF2F$r8AYWAQL$S-NH2 1549.83 775.92 776.15 SP-568Ac-LTF3F$r8AYWAQL$S-NH2 1549.83 775.92 785.12 SP-569Ac-LTF34F2$r8AYWAQL$S-NH2 1567.83 784.92 785.12 SP-570Ac-LTF35F2$r8AYWAQL$S-NH2 1567.83 784.92 1338.74 SP-571Ac-F3CI$r8AYWEAL$A-NH2 1336.66 669.33 705.28 SP-572Ac-F3CI$r8AYWEAL$AA-NH2 1407.70 704.85 680.11 SP-573Ac-F$r8AY6cIWEAL$AA-NH2 1407.70 704.85 736.83 SP-574Ac-F$r8AY6cIWEAL$-NH2 1265.63 633.82 784.1 SP-575Ac-LTF$r8HYWAQLSt/S-NH2 16.03 9.02 826.98 SP-576 Ac-LTF$r8HYWAQL$S-NHsBu1653.93 827.97 828.02 SP-577 Ac-STF$r8AYWAQL$S-NH2 1505.79 753.90 753.94SP-578 Ac-LTF$r8AYWAEL$S-NH2 1532.83 767.42 767.41 SP-579Ac-LTF$r8AYWAQL$E-NH2 1573.85 787.93 787.98 SP-580mdPEG3-LTF$r8AYWAQL$S-NH2 1679.92 840.96 840.97 SP-581Ac-LTF$r8AYWAQhL$S-NH2 1545.86 773.93 774.31 SP-583Ac-LTF$r8AYWAQCha$S-NH2 1571.88 786.94 787.3 SP-584Ac-LTF$r8AYWAQChg$S-NH2 1557.86 779.93 780.4 SP-585Ac-LTF$r8AYWAQCba$S-NH2 1543.84 772.92 780.13 SP-586Ac-LTF$r8AYWAQF$S-NH2 1565.83 783.92 784.2 SP-587Ac-LTF4F$r8HYWAQhL$S-NH2 1629.87 815.94 815.36 SP-588Ac-LTF4F$r8HYWAQCha$S-NH2 1655.89 828.95 828.39 SP-589Ac-LTF4F$r8HYWAQChg$S-NH2 1641.87 821.94 821.35 SP-590Ac-LTF4F$r8HYWAQCba$S-NH2 1627.86 814.93 814.32 SP-591Ac-LTF4F$r8AYWAQhL$S-NH2 1563.85 782.93 782.36 SP-592Ac-LTF4F$r8AYWAQCha$S-NH2 1589.87 795.94 795.38 SP-593Ac-LTF4F$r8AYWAQChg$S-NH2 1575.85 788.93 788.35 SP-594Ac-LTF4F$r8AYWAQCba$S-NH2 1561.83 781.92 781.39 SP-595Ac-LTF3CI$r8AYWAQhL$S-NH2 1579.82 790.91 790.35 SP-596Ac-LTF3CI$r8AYWAQCha$S-NH2 1605.84 803.92 803.67 SP-597Ac-LTF3CI$r8AYWAQChg$S-NH2 1591.82 796.91 796.34 SP-598Ac-LTF3CI$r8AYWAQCba$S-NH2 1577.81 789.91 789.39 SP-599Ac-LTF$r8AYWAQhF$S-NH2 1579.84 790.92 791.14 SP-600Ac-LTF$r8AYWAQF3CF3$S-NH2 1633.82 817.91 818.15 SP-601Ac-LTF$r8AYWAQF3Me$S-NH2 1581.86 791.93 791.32 SP-602Ac-LTF$r8AYWAQ1Nal$S-NH2 1615.84 808.92 809.18 SP-603Ac-LTF$r8AYWAQBip$S-NH2 1641.86 821.93 822.13 SP-604Ac-LTF$r8FYWAQL$A-NH2 1591.88 796.94 797.33 SP-605Ac-LTF$r8HYWAQL$S-NHAm 1667.94 834.97 835.92 SP-606Ac-LTF$r8HYWAQL$S-NHiAm 1667.94 834.97 835.55 SP-607Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1715.94 858.97 859.79 SP-608Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1681.96 841.98 842.49 SP-610Ac-LTF$r8HYWAQL$S-NHnPr 1639.91 820.96 821.58 SP-611Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1707.98 854.99 855.35 SP-612Ac-LTF$r8HYWAQL$S-NHHex 1681.96 841.98 842.4 SP-613Ac-LTF$r8AYWAQL$S-NHmdPeg2 1633.91 817.96 818.35 SP-614Ac-LTF$r8AYWAQL$A-NHmdPeg2 1617.92 809.96 810.3 SP-615Ac-LTF$r8AYWAQL$A-NHmdPeg4 1705.97 853.99 854.33 SP-616Ac-F$r8AYdI4mWEAL$A-NH2 1316.72 659.36 659.44 SP-617Ac-F$r8AYdI5cIWEAL$A-NH2 1336.66 669.33 669.43 SP-618Ac-LThF$r8AYWAQL$S-NH2 1545.86 773.93 774.11 SP-619Ac-LT2Nal$r8AYWAQL$S-NH2 1581.86 791.93 792.43 SP-620Ac-LTA$r8AYWAQL$S-NH2 1455.81 728.91 729.15 SP-621 Ac-LTF$r8AYWVQL$S-NH21559.88 780.94 781.24 SP-622 Ac-LTF$r8HYWAAL$A-NH2 1524.85 763.43 763.86SP-623 Ac-LTF$r8VYWAQL$A-NH2 1543.88 772.94 773.37 SP-624Ac-LTF$r8IYWAQL$S-NH2 1573.89 787.95 788.17 SP-625 Ac-FTF$r8VYWSQL$S-NH21609.85 805.93 806.22 SP-626 Ac-ITF$r8FYWAQL$S-NH2 1607.88 804.94 805.2SP-627 Ac-2NalTF$r8VYWSQL$S-NH2 1659.87 830.94 831.2 SP-628Ac-ITF$r8LYWSQL$S-NH2 1589.89 795.95 796.13 SP-629 Ac-FTF$r8FYWAQL$S-NH21641.86 821.93 822.13 SP-630 Ac-WTF$r8VYWAQL$S-NH2 1632.87 817.44 817.69SP-631 Ac-WTF$r8WYWAQL$S-NH2 1719.88 860.94 861.36 SP-632Ac-VTF$r8AYWSQL$S-NH2 1533.82 767.91 768.19 SP-633 Ac-WTF$r8FYWSQL$S-NH21696.87 849.44 849.7 SP-634 Ac-FTF$r8IYWAQL$S-NH2 1607.88 804.94 805.2SP-635 Ac-WTF$r8VYWSQL$S-NH2 1648.87 825.44 824.8 SP-636Ac-FTF$r8LYWSQL$S-NH2 1623.87 812.94 812.8 SP-637 Ac-YTF$r8FYWSQL$S-NH21673.85 837.93 837.8 SP-638 Ac-LTF$r8AY6cIWEAL$A-NH2 1550.79 776.40776.14 SP-639 Ac-LTF$r8AY6cIWSQL$S-NH2 1581.80 791.90 791.68 SP-640Ac-F$r8AY6cIWSAL$A-NH2 1294.65 648.33 647.67 SP-641Ac-F$r8AY6cIWQAL$AA-NH2 1406.72 704.36 703.84 SP-642Ac-LHF$r8AYWAQL$S-NH2 1567.86 784.93 785.21 SP-643 Ac-LTF$r8AYWAQL$S-NH21531.84 766.92 767.17 SP-644 Ac-LTF$r8AHWAQL$S-NH2 1505.84 753.92 754.13SP-645 Ac-LTF$r8AYWAHL$S-NH2 1540.84 771.42 771.61 SP-646Ac-LTF$r8AYWAQL$H-NH2 1581.87 791.94 792.15 SP-647 H-LTF$r8AYWAQL$A-NH21473.84 737.92 737.29 SP-648 Ac-HHF$r8AYWAQL$S-NH2 1591.83 796.92 797.35SP-649 Ac-aAibWTF$r8VYWSQL$S-NH2 1804.96 903.48 903.64 SP-650Ac-AibWTF$r8HYWAQL$S-NH2 1755.91 878.96 879.4 SP-651Ac-AibAWTF$r8HYWAQL$S-NH2 1826.95 914.48 914.7 SP-652Ac-fWTF$r8HYWAQL$S-NH2 1817.93 909.97 910.1 SP-653Ac-AibWWTF$r8HYWAQL$S-NH2 1941.99 972.00 972.2 SP-654Ac-WTF$r8LYWSQL$S-NH2 1662.88 832.44 832.8 SP-655Ac-WTF$r8NleYWSQL$S-NH2 1662.88 832.44 832.6 SP-656Ac-LTF$r8AYWSQL$a-NH2 1531.84 766.92 767.2 SP-657 Ac-LTF$r8EYWARL$A-NH21601.90 801.95 802.1 SP-658 Ac-LTF$r8EYWAHL$A-NH2 1582.86 792.43 792.6SP-659 Ac-aTF$r8AYWAQL$S-NH2 1489.80 745.90 746.08 SP-660Ac-AibTF$r8AYWAQL$S-NH2 1503.81 752.91 753.11 SP-661Ac-AmfTF$r8AYWAQL$S-NH2 1579.84 790.92 791.14 SP-662Ac-AmwTF$r8AYWAQL$S-NH2 1618.86 810.43 810.66 SP-663Ac-NmLTF$r8AYWAQL$S-NH2 1545.86 773.93 774.11 SP-664Ac-LNmTF$r8AYWAQL$S-NH2 1545.86 773.93 774.11 SP-665Ac-LSarF$r8AYWAQL$S-NH2 1501.83 751.92 752.18 SP-667Ac-LGF$r8AYWAQL$S-NH2 1487.82 744.91 745.15 SP-668Ac-LTNmF$r8AYWAQL$S-NH2 1545.86 773.93 774.2 SP-669 Ac-TF$r8AYWAQL$S-NH21418.76 710.38 710.64 SP-670 Ac-ETF$r8AYWAQL$A-NH2 1531.81 766.91 767.2SP-671 Ac-LTF$r8EYWAQL$A-NH2 1573.85 787.93 788.1 SP-672Ac-LT2Nal$r8AYWSQL$S-NH2 1597.85 799.93 800.4 SP-673Ac-LTF$r8AYWAAL$S-NH2 1474.82 738.41 738.68 SP-674Ac-LTF$r8AYWAQhCha$S-NH2 1585.89 793.95 794.19 SP-675Ac-LTF$r8AYWAQChg$S-NH2 1557.86 779.93 780.97 SP-676Ac-LTF$r8AYWAQCba$S-NH2 1543.84 772.92 773.19 SP-677Ac-LTF$r8AYWAQF3CF3$S-NH2 1633.82 817.91 818.15 SP-678Ac-LTF$r8AYWAQ1Nal$S-NH2 1615.84 808.92 809.18 SP-679Ac-LTF$r8AYWAQBip$S-NH2 1641.86 821.93 822.32 SP-680Ac-LT2Nal$r8AYWAQL$S-NH2 1581.86 791.93 792.15 SP-681Ac-LTF$r8AYWVQL$S-NH2 1559.88 780.94 781.62 SP-682 Ac-LTF$r8AWWAQL$S-NH21554.86 778.43 778.65 SP-683 Ac-FTF$r8VYWSQL$S-NH2 1609.85 805.93 806.12SP-684 Ac-ITF$r8FYWAQL$S-NH2 1607.88 804.94 805.2 SP-685Ac-ITF$r8LYWSQL$S-NH2 1589.89 795.95 796.22 SP-686 Ac-FTF$r8FYWAQL$S-NH21641.86 821.93 822.41 SP-687 Ac-VTF$r8AYWSQL$S-NH2 1533.82 767.91 768.19SP-688 Ac-LTF$r8AHWAQL$S-NH2 1505.84 753.92 754.31 SP-689Ac-LTF$r8AYWAQL$H-NH2 1581.87 791.94 791.94 SP-690 Ac-LTF$r8AYWAHL$S-NH21540.84 771.42 771.61 SP-691 Ac-aAibWTF$r8VYWSQL$S-NH2 1804.96 903.48903.9 SP-692 Ac-AibWTF$r8HYWAQL$S-NH2 1755.91 878.96 879.5 SP-693Ac-AibAWTF$r8HYWAQL$S-NH2 1826.95 914.48 914.7 SP-694Ac-fWTF$r8HYWAQL$S-NH2 1817.93 909.97 910.2 SP-695Ac-AibWWTF$r8HYWAQL$S-NH2 1941.99 972.00 972.7 SP-696Ac-WTF$r8LYWSQL$S-NH2 1662.88 832.44 832.7 SP-697Ac-WTF$r8NleYWSQL$S-NH2 1662.88 832.44 832.7 SP-698Ac-LTF$r8AYWSQL$a-NH2 1531.84 766.92 767.2 SP-699 Ac-LTF$r8EYWARL$A-NH21601.90 801.95 802.2 SP-700 Ac-LTF$r8EYWAHL$A-NH2 1582.86 792.43 792.6SP-701 Ac-aTF$r8AYWAQL$S-NH2 1489.80 745.90 746.1 SP-702Ac-AibTF$r8AYWAQL$S-NH2 1503.81 752.91 753.2 SP-703Ac-AmfTF$r8AYWAQL$S-NH2 1579.84 790.92 791.2 SP-704Ac-AmwTF$r8AYWAQL$S-NH2 1618.86 810.43 810.7 SP-705Ac-NmLTF$r8AYWAQL$S-NH2 1545.86 773.93 774.1 SP-706Ac-LNmTF$r8AYWAQL$S-NH2 1545.86 773.93 774.4 SP-707Ac-LSarF$r8AYWAQL$S-NH2 1501.83 751.92 752.1 SP-708 Ac-TF$r8AYWAQL$S-NH21418.76 710.38 710.8 SP-709 Ac-ETF$r8AYWAQL$A-NH2 1531.81 766.91 767.4SP-710 Ac-LTF$r8EYWAQL$A-NH2 1573.85 787.93 788.2 SP-711Ac-WTF$r8VYWSQL$S-NH2 1648.87 825.44 825.2 SP-713 Ac-YTF$r8FYWSQL$S-NH21673.85 837.93 837.3 SP-714 Ac-F$r8AY6cIWSAL$A-NH2 1294.65 648.33 647.74SP-715 Ac-ETF$r8EYWVQL$S-NH2 1633.84 817.92 817.36 SP-716Ac-ETF$r8EHWAQL$A-NH2 1563.81 782.91 782.36 SP-717 Ac-ITF$r8EYWAQL$S-NH21589.85 795.93 795.38 SP-718 Ac-ITF$r8EHWVQL$A-NH2 1575.88 788.94 788.42SP-719 Ac-ITF$r8EHWAQL$S-NH2 1563.85 782.93 782.43 SP-720Ac-LTF4F$r8AYWAQCba$S-NH2 1561.83 781.92 781.32 SP-721Ac-LTF3CI$r8AYWAQhL$S-NH2 1579.82 790.91 790.64 SP-722Ac-LTF3CI$r8AYWAQCha$S-NH2 1605.84 803.92 803.37 SP-723Ac-LTF3CI$r8AYWAQChg$S-NH2 1591.82 796.91 796.27 SP-724Ac-LTF3CI$r8AYWAQCba$S-NH2 1577.81 789.91 789.83 SP-725Ac-LTF$r8AY6cIWSQL$S-NH2 1581.80 791.90 791.75 SP-726Ac-LTF4F$r8HYWAQhL$S-NH2 1629.87 815.94 815.36 SP-727Ac-LTF4F$r8HYWAQCba$S-NH2 1627.86 814.93 814.32 SP-728Ac-LTF4F$r8AYWAQhL$S-NH2 1563.85 782.93 782.36 SP-729Ac-LTF4F$r8AYWAQChg$S-NH2 1575.85 788.93 788.35 SP-730Ac-ETF$r8EYWVAL$S-NH2 1576.82 789.41 788.79 SP-731 Ac-ETF$r8EHWAAL$A-NH21506.79 754.40 754.8 SP-732 Ac-ITF$r8EYWAAL$S-NH2 1532.83 767.42 767.75SP-733 Ac-ITF$r8EHWVAL$A-NH2 1518.86 760.43 760.81 SP-734Ac-ITF$r8EHWAAL$S-NH2 1506.82 754.41 754.8 SP-735 Pam-LTF$r8EYWAQL$S-NH21786.07 894.04 894.48 SP-736 Pam-ETF$r8EYWAQL$S-NH2 1802.03 902.02902.34 SP-737 Ac-LTF$r8AYWLQL$S-NH2 1573.89 787.95 787.39 SP-738Ac-LTF$r8EYWLQL$S-NH2 1631.90 816.95 817.33 SP-739 Ac-LTF$r8EHWLQL$S-NH21605.89 803.95 804.29 SP-740 Ac-LTF$r8VYWAQL$S-NH2 1559.88 780.94 781.34SP-741 Ac-LTF$r8AYWSQL$S-NH2 1547.84 774.92 775.33 SP-742Ac-ETF$r8AYWAQL$S-NH2 1547.80 774.90 775.7 SP-743 Ac-LTF$r8EYWAQL$S-NH21589.85 795.93 796.33 SP-744 Ac-LTF$r8HYWAQL$S-NHAm 1667.94 834.97835.37 SP-745 Ac-LTF$r8HYWAQL$S-NHiAm 1667.94 834.97 835.27 SP-746Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1715.94 858.97 859.42 SP-747Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1681.96 841.98 842.67 SP-748Ac-LTF$r8HYWAQL$S-NHnBu 1653.93 827.97 828.24 SP-749Ac-LTF$r8HYWAQL$S-NHnPr 1639.91 820.96 821.31 SP-750Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1707.98 854.99 855.35 SP-751Ac-LTF$r8HYWAQL$S-NHHex 1681.96 841.98 842.4 SP-752Ac-LTF$r8AYWAQL$S-NHmdPeg2 1633.91 817.96 855.35 SP-753Ac-LTF$r8AYWAQL$A-NHmdPeg2 1617.92 809.96 810.58 SP-754Ac-LTF$r5AYWAAL$s8S-NH2 1474.82 738.41 738.79 SP-755Ac-LTF$r8AYWCouQL$S-NH2 1705.88 853.94 854.61 SP-756Ac-LTF$r8CouYWAQL$S-NH2 1705.88 853.94 854.7 SP-757Ac-CouTF$r8AYWAQL$S-NH2 1663.83 832.92 833.33 SP-758H-LTF$r8AYWAQL$A-NH2 1473.84 737.92 737.29 SP-759 Ac-HHF$r8AYWAQL$S-NH21591.83 796.92 797.72 SP-760 Ac-LT2Nal$r8AYWSQL$S-NH2 1597.85 799.93800.68 SP-761 Ac-LTF$r8HCouWAQL$S-NH2 1679.87 840.94 841.38 SP-762Ac-LTF$r8AYWCou2QL$S-NH2 1789.94 895.97 896.51 SP-763Ac-LTF$r8Cou2YWAQL$S-NH2 1789.94 895.97 896.5 SP-764Ac-Cou2TF$r8AYWAQL$S-NH2 1747.90 874.95 875.42 SP-765Ac-LTF$r8ACou2WAQL$S-NH2 1697.92 849.96 850.82 SP-766Dmaac-LTF$r8AYWAQL$S-NH2 1574.89 788.45 788.82 SP-767Hexac-LTF$r8AYWAQL$S-NH2 1587.91 794.96 795.11 SP-768Napac-LTF$r8AYWAQL$S-NH2 1657.89 829.95 830.36 SP-769Pam-LTF$r8AYWAQL$S-NH2 1728.06 865.03 865.45 SP-770Ac-LT2Nal$r8HYAAQL$S-NH2 1532.84 767.42 767.61 SP-771Ac-LT2Nal$/r8HYWAQL$/S-NH2 1675.91 838.96 839.1 SP-772Ac-LT2Nal$r8HYFAQL$S-NH2 1608.87 805.44 805.9 SP-773Ac-LT2Nal$r8HWAAQL$S-NH2 1555.86 778.93 779.08 SP-774Ac-LT2Nal$r8HYAWQL$S-NH2 1647.88 824.94 825.04 SP-775Ac-LT2Nal$r8HYAAQW$S-NH2 1605.83 803.92 804.05 SP-776Ac-LTW$r8HYWAQL$S-NH2 1636.88 819.44 819.95 SP-777Ac-LT1Nal$r8HYWAQL$S-NH2 1647.88 824.94 825.41

In the sequences shown above and elsewhere, the following abbreviationsare used: “Nle” represents norleucine, “Aib” represents2-aminoisobutyric acid, “Ac” represents acetyl, and “Pr” representspropionyl Amino acids represented as “$” are alpha-MeS5-pentenyl-alanine olefin amino acids connected by an all-carbon i toi+4 crosslinker comprising one double bond Amino acids represented as“$r5” are alpha-Me R5-pentenyl-alanine olefin amino acids connected byan all-carbon i to i+4 crosslinker comprising one double bond Aminoacids represented as “$s8” are alpha-Me S8-octenyl-alanine olefin aminoacids connected by an all-carbon i to i+7 crosslinker comprising onedouble bond Amino acids represented as “$r8” are alpha-MeR8-octenyl-alanine olefin amino acids connected by an all-carbon i toi+7 crosslinker comprising one double bond. “Ahx” represents anaminocyclohexyl linker. The crosslinkers are linear all-carboncrosslinker comprising eight or eleven carbon atoms between the alphacarbons of each amino acid Amino acids represented as “$/” are alpha-MeS5-pentenyl-alanine olefin amino acids that are not connected by anycrosslinker Amino acids represented as “$/r5” are alpha-MeR5-pentenyl-alanine olefin amino acids that are not connected by anycrosslinker. Amino acids represented as “$/s8” are alpha-MeS8-octenyl-alanine olefin amino acids that are not connected by anycrosslinker Amino acids represented as “$/r8” are alpha-MeR8-octenyl-alanine olefin amino acids that are not connected by anycrosslinker Amino acids represented as “Amw” are alpha-Me tryptophanamino acids Amino acids represented as “Aml” are alpha-Me leucine aminoacids Amino acids represented as “2ff” are 2-fluoro-phenylalanine aminoacids. Amino acids represented as “3ff” are 3-fluoro-phenylalanine aminoacids. Amino acids represented as “St” are amino acids comprising twopentenyl-alanine olefin side chains, each of which is crosslinked toanother amino acid as indicated Amino acids represented as “SO” areamino acids comprising two pentenyl-alanine olefin side chains that arenot crosslinked Amino acids represented as “% St” are amino acidscomprising two pentenyl-alanine olefin side chains, each of which iscrosslinked to another amino acid as indicated via fully saturatedhydrocarbon crosslinks.

For example, the compounds represented as SP-72, SP-56 and SP-138 havethe following structures:

For example, additional compounds have the following structures:

Example 3 Competition Binding ELISA (HDM2 & HDMX)

p53-His6 protein (30 nM/well) is coated overnight at room temperature inthe wells of a 96-well Immulon plates. On the day of the experiment,plates are washed with 1×PBS-Tween 20 (0.05%) using an automated ELISAplate washer, blocked with ELISA Micro well Blocking for 30 minutes atroom temperature; excess blocking agent is washed off by washing plateswith 1×PBS-Tween 20 (0.05%). Peptides are diluted from 10 mM DMSO stocksto 500 μM working stocks in sterile water, further dilutions made in0.5% DMSO to keep the concentration of DMSO constant across the samples.The peptides are added to wells at 2× desired concentrations in 50 μlvolumes, followed by addition of diluted GST-HDM2 or GST-HMDX protein(final concentration: 10 nM). Samples are incubated at room temperaturefor 2 h, plates are washed with PBS-Tween 20 (0.05%) prior to adding 100μl of HRP-conjugated anti-GST antibody [Hypromatrix, INC] diluted to 0.5μg/ml in HRP-stabilizing buffer. Post 30 min incubation with detectionantibody, plates are washed and incubated with 100 μl per well of TMB-ESubstrate solution up to 30 minutes; reactions are stopped using 1M HCLand absorbance measured at 450 nm on micro plate reader. Data isanalyzed using Graph Pad PRISM software.

Example 4 SJSA-1 Cell Viability Assay

SJSA1 cells are seeded at the density of 5000 cells/100 μl/well in96-well plates a day prior to assay. On the day of study cells arewashed once with Opti-MEM Media and 90 μL of the Opti-MEM Media is addedto cells. Peptides are diluted from 10 mM DMSO stocks to 500 μM workingstocks in sterile water, further dilutions made in 0.5% DMSO to keep theconcentration of DMSO constant across the samples. The finalconcentration range μM will be 50, 25, 12.5, 6.25, 3.1, 1.56, 0.8 and 0μM in 100 μL final volume per well for peptides. Final highest DMSOconcentration is 0.5% and will be used as the negative control. CaymanChemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positivecontrol. Nutlin was diluted using the same dilution scheme as peptides10 μl of 10× desired concentrations is added to the appropriate well toachieve the final desired concentrations. Cells are then incubated withpeptides for 20-24 h at 37° C. in humidified 5% CO2 atmosphere.Post-incubation period, cell viability is measured using Promega CellTiter-Glo reagents according to manufacturer' instructions.

Example 5 SJSA-1 p21 Up-Regulation Assay

SJSA1 cells are seeded at the density of 0.8 million cells/2 ml/well in6-well plates a day prior to assay. On the day of study cells are washedonce with Opti-MEM Media and 1350 μL of the Opti-MEM Media is added tocells. Peptides are diluted from 10 mM DMSO stocks to 500 μM workingstocks in sterile water, further dilutions made in 0.5% DMSO to keep theconcentration of DMSO constant across the samples. Final highest DMSOconcentration is 0.5% and is used as the negative control. CaymanChemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positivecontrol. Nutlin is diluted using the same dilution scheme as peptides150 μl of 10× desired concentrations is added to the appropriate well toachieve the final desired concentrations. Cells are then incubated withpeptides for 18-20 h at 37° C. in humidified 5% CO2 atmosphere.Post-incubation period, cells are harvested, washed with 1×PBS (withoutCa++/Mg++) and lysed in 1× Cell lysis buffer (Cell Signalingtechnologies 10× buffer diluted to 1× and supplemented with proteaseinhibitors and Phosphatase inhibitors) on ice for 30 min. Lysates arecentrifuged at 13000 rpm speed in a microfuge at 40 C for 8 min; clearsupernatants are collected and stored at −80° C. till further use. Totalprotein content of the lysates is measured using BCA protein detectionkit and BSA standards from Thermofisher. 25 μg of the total protein isused for p21 detection ELISA assay. Each condition is set in triplicatefor ELISA plate. The ELISA assay protocol is followed as per themanufacturer's instructions. 25 μg total protein used for each well, andeach well is set up in triplicate. Data is analyzed using Graph PadPRISM software.

Example 6 p53 GRIP Assay

Thermo Scientific* Biolmage p53-Hdm2 Redistribution Assay monitors theprotein interaction with Hdm2 and cellular translocation of GFP-taggedp53 in response to drug compounds or other stimuli. Recombinant CHO-hIRcells stably express human p53(1-312) fused to the C-terminus ofenhanced green fluorescent protein (EGFP) and PDE4A4-Hdm2(1-124), afusion protein between PDE4A4 and Hdm2(1-124). They provide aready-to-use assay system for measuring the effects of experimentalconditions on the interaction of p53 and Hdm2. Imaging and analysis isperformed with a HCS platform.

CHO-hIR cells are regularly maintained in Ham's F12 media supplementedwith 1% Penicillin-Streptomycin, 0.5 mg/ml Geneticin, 1 mg/ml Zeocin and10% FBS. Cells seeded into 96-well plates at the density of 7000cells/100 μl per well 18-24 hours prior to running the assay usingculture media. The next day, media is refreshed and PD177 is added tocells to the final concentration of 3 μM to activate foci formation.Control wells are kept without PD-177 solution. 24 h post stimulationwith PD177, cells are washed once with Opti-MEM Media and 50 μL of theOpti-MEM Media supplemented with PD-177(6 μM) is added to cells.Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks insterile water, further dilutions made in 0.5% DMSO to keep theconcentration of DMSO constant across the samples. Final highest DMSOconcentration is 0.5% and is used as the negative control. CaymanChemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positivecontrol. Nutlin was diluted using the same dilution scheme as peptides.50 μl of 2× desired concentrations is added to the appropriate well toachieve the final desired concentrations. Cells are then incubated withpeptides for 6 h at 37° C. in humidified 5% CO2 atmosphere.Post-incubation period, cells are fixed by gently aspirating out themedia and adding 150 μl of fixing solution per well for 20 minutes atroom temperature. Fixed cells are washed 4 times with 200 μl PBS perwell each time. At the end of last wash, 100 μl of 1 μM Hoechst stainingsolution is added. Sealed plates incubated for at least 30 min in dark,washed with PBS to remove excess stain and PBS is added to each well.Plates can be stored at 4° C. in dark up to 3 days. The translocation ofp53/HDM2 is imaged using Molecular translocation module on CellomicsArrayscan instrument using 10× objective, XF-100 filter sets for Hoechstand GFP. The output parameters was Mean−CircRINGAveIntenRatio (the ratioof average fluorescence intensities of nucleus and cytoplasm, (wellaverage)). The minimally acceptable number of cells per well used forimage analysis was set to 500 cells.

Example 7 Direct Binding Assay hDM2 with Fluorescence Polarization (FP)

The assay was performed according to the following general protocol:

-   -   1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt        buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 μM working        stock solution.    -   2. Add 30 μl of 10 μM of protein stock solution into A1 and B1        well of 96-well black HE microplate (Molecular Devices).    -   3. Fill in 30 μl of FP buffer into column A2 to A12, B2 to B12,        C1 to C12, and D1 to D12.    -   4. 2 or 3 fold series dilution of protein stock from A1, B1 into        A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM        concentration at the last dilution point.    -   5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with        DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10        μM with water (dilution 1:10) and then dilute with FP buffer        from 10 μM to 40 nM (dilution 1:250). This is the working        solution which will be a 10 nM concentration in well (dilution        1:4). Keep the diluted FAM labeled peptide in the dark until        use.    -   6. Add 10 μl of 10 nM of FAM labeled peptide into each well and        incubate, and read at different time points. Kd with        5-FAM-BaLTFEHYWAQLTS-NH₂ is ˜13.38 nM.

Example 8 Competitive Fluorescence Polarization Assay for hDM2

The assay was performed according to the following general protocol:

-   -   1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt        buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 84 nM (2×)        working stock solution.    -   2. Add 20 μl of 84 nM (2×) of protein stock solution into each        well of 96-well black HE microplate (Molecular Devices)    -   3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with        DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10        μM with water (dilution 1:10) and then dilute with FP buffer        from 10 μM to 40 nM (dilution 1:250). This is the working        solution which will be a 10 nM concentration in well (dilution        1:4). Keep the diluted FAM labeled peptide in the dark until        use.    -   4. Make unlabeled peptide dose plate with FP buffer starting        with 1 μM (final) of peptide and making 5 fold serial dilutions        for 6 points using following dilution scheme.    -   Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2).        Then, dilute from 5 mM to 500 μM with H₂O (dilution 1:10) and        then dilute with FP buffer from 500 μM to 20 μM (dilution 1:25).        Making 5 fold serial dilutions from 4 μM (4×) for 6 points.    -   5. Transfer 10 μl of serial diluted unlabeled peptides to each        well which is filled with 20 μl of 84 nM of protein.    -   6. Add 10 μl of 10 nM (4×) of FAM labeled peptide into each well        and incubate for 3 hr to read.

Results of Examples 7 and 8 are provided in HDM2 data in FIGS. 7A-D.

Example 9 Direct Binding Assay hDMX with Fluorescence Polarization (FP)

The assay was performed according to the following general protocol:

-   -   1. Dilute hDMX (In-house, 40 kD) into FP buffer (High salt        buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 μM working        stock solution.    -   2. Add 30 μl of 10 μM of protein stock solution into A1 and B1        well of 96-well black HE microplate (Molecular Devices).    -   3. Fill in 30 μl of FP buffer into column A2 to A12, B2 to B12,        C1 to C12, and D1 to D12.    -   4. 2 or 3 fold series dilution of protein stock from A1, B1 into        A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM        concentration at the last dilution point.    -   5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with        DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10        μM with water (dilution 1:10) and then dilute with FP buffer        from 10 μM to 40 nM (dilution 1:250). This is the working        solution which will be a 10 nM concentration in well (dilution        1:4). Keep the diluted FAM labeled peptide in the dark until        use.    -   6. Add 10 μl of 10 nM of FAM labeled peptide into each well and        incubate, and read at different time points.    -   Kd with 5-FAM-BaLTFEHYWAQLTS-NH₂ is ˜51 nM.

Example 10 Competitive Fluorescence Polarization Assay for hDMX

The assay was performed according to the following general protocol:

-   -   1. Dilute hDMX (In-house, 40 kD) into FP buffer (High salt        buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5.) to make 300 nM (2×)        working stock solution.    -   2. Add 20 μl of 300 nM (2×) of protein stock solution into each        well of 96-well black HE microplate (Molecular Devices)    -   3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with        DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10        μM with water (dilution 1:10) and then dilute with FP buffer        from 10 μM to 40 nM (dilution 1:250). This is the working        solution which will be a 10 nM concentration in well (dilution        1:4). Keep the diluted FAM labeled peptide in the dark until        use.    -   4. Make unlabeled peptide dose plate with FP buffer starting        with 5 μM (final) of peptide and making 5 fold serial dilutions        for 6 points using following dilution scheme.    -   5. Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2).        Then, dilute from 5 mM to 500 μM with H₂O (dilution 1:10) and        then dilute with FP buffer from 500 μM to 20 μM (dilution 1:25).        Making 5 fold serial dilutions from 20 μM (4×) for 6 points.    -   6. Transfer 10 μl of serial diluted unlabeled peptides to each        well which is filled with 20 μl of 300 nM of protein.    -   7. Add 10 μl of 10 nM (4×) of FAM labeled peptide into each well        and incubate for 3 hr to read.

Results of Examples 9 and 10 are provided in HDMX data in FIGS. 7A-D.

Example 11 Cell Viability Assay

The assay was performed according to the following general protocol:

Cell Plating: Trypsinize, count and seed cells at the pre-determineddensities in 96-well plates a day prior to assay. Following celldensities are used for each cell line in use:

-   -   SJSA-1: 7500 cells/well    -   RKO: 5000 cells/well    -   RK0-E6: 5000 cells/well    -   HCT-116: 5000 cells/well    -   SW-480: 2000 cells/well    -   MCF-7: 5000 cells/well

On the day of study, replace media with fresh media with 11% FBS (assaymedia) at room temperature. Add 180 μL of the assay media per well.Control wells with no cells, receive 200 μl media.

Peptide dilution: all dilutions are made at room temperature and addedto cells at room temperature.

-   -   Prepare 10 mM stocks of the peptides in DMSO. Serially dilute        the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33,        0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the        serially DMSO-diluted peptides 33.3 times using sterile water.        This gives range of 10× working stocks. Also prepare        DMSO/sterile water (3% DMSO) mix for control wells.    -   Thus the working stocks concentration range μM will be 300, 100,        30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using        multichannel.    -   Row H has controls. H1-H3 will receive 20 ul of assay media.        H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will        have media alone control with no cells.    -   Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10        mM) is used as positive control. Nutlin was diluted using the        same dilution scheme as peptides.

Addition of Working Stocks to Cells:

-   -   Add 20 μl of 10× desired concentration to appropriate well to        achieve the final concentrations in total 200 μl volume in well.        (20 μl of 300 μM peptide+180 μl of cells in media=30 μM final        concentration in 200 μl volume in wells). Mix gently a few times        using pipette. Thus final concentration range used will be 30,        10, 3, 1, 0.3, 0.1, 0.03 & 0 μM (for potent peptides further        dilutions are included).    -   Controls include wells that get no peptides but contain the same        concentration of DMSO as the wells containing the peptides, and        wells containing NO CELLS.    -   Incubate for 72 hours at 37° C. in humidified 5% CO₂ atmosphere.    -   The viability of cells is determined using MTT reagent from        Promega. Viability of SJSA-1, RKO, RKO-E6, HCT-116 cells is        determined on day 3, MCF-7 cells on day 5 and SW-480 cells on        day 6. At the end of designated incubation time, allow the        plates to come to room temperature. Remove 80 μl of assay media        from each well. Add 15 μl of thawed MTT reagent to each well.    -   Allow plate to incubate for 2 h at 37° C. in humidified 5% CO₂        atmosphere and add 100 μl solubilization reagent as per        manufacturer's protocol. Incubate with agitation for 1 h at room        temperature and read on Synergy Biotek multiplate reader for        absorbance at 570 nM.    -   Analyze the cell viability against the DMSO controls using        GraphPad PRISM analysis tools.

Reagents:

-   -   Invitrogen cell culture Media        -   i. Falcon 96-well clear cell culture treated plates (Nunc            353072)    -   DMSO (Sigma D 2650)    -   RPMI 1640 (Invitrogen 72400)    -   MTT (Promega G4000)

Instruments:

Multiplate Reader for Absorbance readout (Synergy 2)

Results of Example 11 are provided in SJSA-1 EC50 data in FIGS. 7A-D.

Example 12 P21 ELISA Assay

The assay was performed according to the following general protocol:

Cell Plating:

-   -   Trypsinize, count and seed SJSA1 cells at the density of 7500        cells/100 μl/well in 96-well plates a day prior to assay.    -   On the day of study, replace media with fresh RPMI-11% FBS        (assay media). Add 90 μL of the assay media per well. Control        wells with no cells, receive 100 μl media.

Peptide Dilution:

-   -   Prepare 10 mM stocks of the peptides in DMSO. Serially dilute        the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33,        0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the        serially DMSO-diluted peptides 33.3 times using sterile water        This gives range of 10× working stocks. Also prepare        DMSO/sterile water (3% DMSO) mix for control wells.    -   Thus the working stocks concentration range μM will be 300, 100,        30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using        multichannel.    -   Row H has controls. H1-H3 will receive 10 ul of assay media.        H4-H9 will receive 10 ul of 3% DMSO-water vehicle. H10-H12 will        have media alone control with no cells.    -   Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10        mM) is used as positive control. Nutlin was diluted using the        same dilution scheme as peptides.

Addition of Working Stocks to Cells:

-   -   Add 10 μl of 10× desired concentration to appropriate well to        achieve the final concentrations in total 100 μl volume in well.        (10 μl of 300 μM peptide+90 μl of cells in media=30 μM final        concentration in 100 μl volume in wells). Thus final        concentration range used will be 30, 10, 3, 1, 0.3& 0 μM.    -   Controls will include wells that get no peptides but contain the        same concentration of DMSO as the wells containing the peptides,        and wells containing NO CELLS.    -   20 h-post incubation, aspirate the media; wash cells with 1×PBS        (without Ca⁺⁺/Mg⁺⁺) and lyse in 60 μl of 1× Cell lysis buffer        (Cell Signaling technologies 10× buffer diluted to 1× and        supplemented with protease inhibitors and Phosphatase        inhibitors) on ice for 30 min.    -   Centrifuge plates in at 5000 rpm speed in at 4° C. for 8 min;        collect clear supernatants and freeze at −80° C. till further        use.

Protein Estimation:

-   -   Total protein content of the lysates is measured using BCA        protein detection kit and BSA standards from Thermofisher.        Typically about 6-7 μg protein is expected per well.    -   Use 50 μl of the lysate per well to set up p21 ELISA.

Human Total p21 ELISA:

The ELISA assay protocol is followed as per the manufacturer'sinstructions. 50 μl lysate is used for each well, and each well is setup in triplicate.

Reagents:

-   -   Cell-Based Assay (−)-Nutlin-3 (10 mM): Cayman Chemicals, catalog        #600034    -   OptiMEM, Invitrogen catalog #51985    -   Cell Signaling Lysis Buffer (10×), Cell signaling technology,        Catalog #9803    -   Protease inhibitor Cocktail tablets(mini), Roche Chemicals,        catalog #04693124001    -   Phosphatase inhibitor Cocktail tablet, Roche Chemicals, catalog        #04906837001    -   Human total p21 ELISA kit, R&D Systems, DYC1047-5    -   STOP Solution (1M HCL), Cell Signaling Technologies, Catalog        #7002

Instruments: Micro centrifuge—Eppendorf 5415D and Multiplate Reader forAbsorbance readout (Synergy 2)

Results of Example 12 are provided in p21 data in FIGS. 7A-D.

Example 13 Caspase 3 Detection Assay

The assay was performed according to the following general protocol:

Cell Plating:

Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/100μl/well in 96-well plates a day prior to assay. On the day of study,replace media with fresh RPMI-11% FBS (assay media). Add 180 μL of theassay media per well. Control wells with no cells, receive 200 μl media.

Peptide Dilution:

-   -   Prepare 10 mM stocks of the peptides in DMSO. Serially dilute        the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33,        0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the        serially DMSO-diluted peptides 33.3 times using sterile water        This gives range of 10× working stocks. Also prepare        DMSO/sterile water (3% DMSO) mix for control wells.    -   Thus the working stocks concentration range μM will be 300, 100,        30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using        multichannel. Add 20 ul of 10× working stocks to appropriate        wells.    -   Row H has controls. H1-H3 will receive 20 ul of assay media.        H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will        have media alone control with no cells.    -   Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10        mM) is used as positive control. Nutlin was diluted using the        same dilution scheme as peptides.

Addition of Working Stocks to Cells:

-   -   Add 10 μl of 10× desired concentration to appropriate well to        achieve the final concentrations in total 100 μl volume in well.        (10 μl of 300 μM peptide+90 μl of cells in media=30 μM final        concentration in 100 μl volume in wells). Thus final        concentration range used will be 30, 10, 3, 1, 0.3& 0 μM.    -   Controls will include wells that get no peptides but contain the        same concentration of DMSO as the wells containing the peptides,        and wells containing NO CELLS.    -   48 h-post incubation, aspirate 80 μl media from each well; add        100 μl Caspase 3/7Glo assay reagent (Promega Caspase 3/7 glo        assay system, G8092)per well, incubate with gentle shaking for 1        h at room temperature.    -   read on Synergy Biotek multiplate reader for luminescence.    -   Data is analyzed as Caspase 3 activation over DMSO-treated        cells.

Results of Example 13 are provided in p21 data in FIGS. 7A-D.

1. A peptidomimetic macrocycle comprising an amino acid sequence whichis at least about 60% identical to an amino acid sequence chosen fromthe group consisting of the amino acid sequences in Table 1, 2, 3, or 4.2. The peptidomimetic macrocycle of claim 1, wherein the amino acidsequence of said peptidomimetic macrocycle is at least about 60%identical to an amino acid sequence chosen from the group consisting ofthe amino acid sequences in Table 1, 2, 3, or 4, and the macrocycle doesnot contain a triazole or a thioether.
 3. The peptidomimetic macrocycleof claim 1, wherein the amino acid sequence of said peptidomimeticmacrocycle is at least about 80% identical to an amino acid sequencechosen from the group consisting of the amino acid sequences in Table 1,2, 3, or
 4. 4. The peptidomimetic macrocycle of claim 1, wherein theamino acid sequence of said peptidomimetic macrocycle is at least about90% identical to an amino acid sequence chosen from the group consistingof the amino acid sequences in Table 1, 2, 3, or
 4. 5. Thepeptidomimetic macrocycle of claim 1, wherein the amino acid sequence ofsaid peptidomimetic macrocycle is chosen from the group consisting ofthe amino acid sequences in Table 1, 2, 3, or
 4. 6. The peptidomimeticmacrocycle of claim 1, wherein the peptidomimetic macrocycle comprises ahelix.
 7. The peptidomimetic macrocycle of claim 1, wherein thepeptidomimetic macrocycle comprises an α-helix.
 8. The peptidomimeticmacrocycle of claim 1, wherein the peptidomimetic macrocycle comprisesan α,α-disubstituted amino acid.
 9. The peptidomimetic macrocycle ofclaim 1, wherein the peptidomimetic macrocycle comprises a crosslinkerlinking the α-positions of at least two amino acids.
 10. Thepeptidomimetic macrocycle of claim 9, wherein at least one of said twoamino acids is an α,α-disubstituted amino acid.
 11. The peptidomimeticmacrocycle of claim 1, wherein the peptidomimetic macrocycle has theformula:

wherein: each A, C, D, and E is independently a natural or non-naturalamino acid, and D and E independently optionally include a cappinggroup; B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO-], [—NH-L₃-SO₂—], or [—NH-L₃-]; R₁ and R₂ are independently—H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl, unsubstituted or substituted withhalo-; R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅; L is amacrocycle-forming linker of the formula -L₁-L₂-; L₁ and L₂ areindependently alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₄—K—R₄—]_(n), each being optionally substituted with R₅; each R₄ isalkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene; each K is O, S, SO, SO₂,CO, CO₂, or CONR₃; each R₅ is independently halogen, alkyl, —OR₆,—N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, aradioisotope or a therapeutic agent; each R₆ is independently —H, alkyl,alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, afluorescent moiety, a radioisotope or a therapeutic agent; R₇ is —H,alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue; R₉ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue; v and w are independently integers from1-1000; u is an integer from 1-10; x, y and z are independently integersfrom 0-10; and n is an integer from 1-5.
 12. The peptidomimeticmacrocycle of claim 11, wherein L does not include a thioether or atriazole.
 13. The peptidomimetic macrocycle of claim 1, wherein thepeptidomimetic macrocycle comprises a crosslinker linking a backboneamino group of a first amino acid to a second amino acid within thepeptidomimetic macrocycle.
 14. The peptidomimetic macrocycle of claim13, wherein the peptidomimetic macrocycle has the formula (IV) or (IVa):

wherein: each A, C, D, and E is independently a natural or non-naturalamino acid, and D and E independently optionally include a cappinggroup; B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO-], [—NH-L₃-SO₂—], or [—NH-L₃-]; R₁ and R₂ are independently—H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl, unsubstituted or substituted withhalo-, or part of a cyclic structure with an E residue; R₃ is hydrogen,alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅; L₁ and L₂ are independently alkylene,alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₄—K—R₄—]_(n), each being optionally substituted with R₅; each R₄ isalkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene; each K is O, S, SO, SO₂,CO, CO₂, or CONR₃; each R₅ is independently halogen, alkyl, —OR₆,—N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, aradioisotope or a therapeutic agent; each R₆ is independently —H, alkyl,alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, afluorescent moiety, a radioisotope or a therapeutic agent; R₇ is —H,alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅; v and w are independently integers from1-1000; u is an integer from 1-10; x, y and z are independently integersfrom 0-10; and n is an integer from 1-5.
 15. The peptidomimeticmacrocycle of claim 14, wherein L₁ and L₂ either alone or in combinationdo not include a thioether or a triazole.
 16. The peptidomimeticmacrocycle of claim 11, wherein L₁ and L₂ are independently alkylene,alkenylene or alkynylene.
 17. The peptidomimetic macrocycle of claim 11,wherein L₁ and L₂ are independently C₃-C₁₀ alkylene or alkenylene. 18.The peptidomimetic macrocycle of claim 17, wherein L₁ and L₂ areindependently C₃-C₆ alkylene or alkenylene.
 19. The peptidomimeticmacrocycle of claim 11, wherein R₁ and R₂ are H.
 20. The peptidomimeticmacrocycle of claim 11, wherein R₁ and R₂ are independently alkyl. 21.The peptidomimetic macrocycle of claim 11, wherein R₁ and R₂ are methyl.22. A method of treating cancer in a subject comprising administering tothe subject a peptidomimetic macrocycle of claim
 1. 23. A method ofmodulating the activity of p53 and/or HDM2 and/or HDMX in a subjectcomprising administering to the subject a peptidomimetic macrocycle ofclaim
 1. 24. A method of antagonizing the interaction between p53 andHDM2 and/or between p53 and HDMXproteins in a subject comprisingadministering to the subject a peptidomimetic macrocycle of claim 1.